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Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses
Vaccine 19 (2001) 2329– 2336 www.elsevier.com/locate/vaccine
Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses Z. Wang, L. Hangartner, T.I. Cornu, L.R. Martin, A. Zuniga, M.A. Billeter *, H.Y. Naim Institute of Molecular Biology, Uni6ersity of Zurich-Irchel, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Abstract We have genetically engineered a panel of recombinant measles viruses (rMVs) that express from various positions within the MV genome either the HN or F surface glycoproteins of mumps virus (MuV) or the env, gag or pol proteins from simian immunodeficiency virus (SIV). All rMVs were rescued from the respective antigenomic plasmid constructs; progeny viruses replicated comparably to the progenitor Edmonston B MV, but showed slight propagation retardation, which was dependent on the size and nature of the expressed proteins and on the genomic position of the inserts. All transgenes except that encoding mumps F glycoprotein were faithfully maintained and expressed even after virus amplification by 1020. Our results suggest possible applications of rMVs as live-attenuated, multivalent vaccines against retroviruses such as SIV and HIV as well as other pathogens more distantly related to MV than MuV. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Attenuated measles viruses; Live vaccines; Expression vectors
1. Introduction Measles virus (MV), an enveloped virus with a nonsegmented, tightly encapsidated negative-strand RNA genome, belongs to the family Paramyxo6iride, genus Morbilli6irus [1,2]. MV cDNA plasmids have been constructed to produce precisely initiated and terminated MV antigenomes related to the Edmonston B vaccine strains, and a helper-cell-based system has been established which allows the rescue of MV from such MV antigenomic plasmids [3]. This allows the construction of genetically altered MV and of recombinant derivatives of MV (rMVs) expressing proteins from inserted transgenes [4,5]. The most complex rMV generated so far, exceeding in length the standard MV by more than 5000 nucleotides, contains the coding regions for green fluorescent protein (GFP), beta-galactosidase (b-gal) and chloramphenicol acetyl transferase (CAT), engineered into three positions of the MV antigenome; the inserted coding regions are expressed as independent transcription units [4]. These transgenes were expressed * Corresponding author. Tel.: +41-1-6353123; fax: +41-16356864. E-mail address: [email protected] (M.A. Billeter).
at levels, according to their genomic position, and were stably maintained over many viral generations. This is rather surprising for an RNA virus-based vector, since all RNA polymerases are error-prone, and positivestrand RNA viruses are notorious for spontaneous deletion of inserted genetic material, presumably by nonhomologous copy-choice recombination events. Recombinant MVs expressing biologically relevant proteins such as IL-12 or small proteins derived from other pathogens, including the small surface protein and the core protein of hepatitis B virus (HBV), have shown only minor propagation delays and little impairment of end titers [6,7]. In addition, the complete replacement of the MV envelope proteins F and H by the single envelope protein G of the distantly related vesicular stomatitis virus (VSV) generated stable chimeric viruses (MV/VSV) that gained the tropism of VSV [5]. The ability to construct new recombinant and chimeric MVs opens the prospect to develop new vaccines based on MV. The limited immunization experiments carried out so far with rMVs have been encouraging. The MV expressing HBV antigens induced vigorous humoral immune responses in transgenic mice, which have been made susceptible to MV
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replication by insertion of a YAC encompassing the gene encoding the MV receptor CD46 and elimination of the interferon type 1 receptor [8,9]. Squirrel monkeys Saimiri inoculated with b-gal-expressing MV generated both strong humoral and cellular immune responses against b-gal, (F. Tangy, pers. commun.), although b-gal is inherently not a potent immunogen. Interferon defective mice were protected against lethal doses of VSV by immunization with the MV-VSV chimera [10]. In comparison with other viral vector systems in use for the delivery of foreign proteins and for immunization purposes, the MV Edmonston vaccine strain-based vector system has decisive advantages. The Edmonston B strain has a long history as a safe and efficacious vaccine in human subjects, inducing life-long protection against measles usually by a single application; furthermore, the production cost of vaccination doses is very low. It may be expected that rMVs can induce longlasting immunity both against MV and the pathogens from which the inserted transgenes are derived. In the present study, we investigated the feasibility to generate rMVs expressing transgenes derived either from the mumps virus (MuV), which is relatively closely related to MV, or simian immunodeficiency virus (SIV), to see whether the expression of large viral components strongly interferes with the propagation of the vector. In contrast to one MV/MuV recombinant,
all MV/SIV recombinants turned out to stably maintain the expression of the inserted reading frames. The latter recombinants are of particular interest; if immunization of macaques with a mixture of these and other recombinants expressing SIV tat and nef (F. Tangy, unpublished results) will induce both strong humoral and cell-mediated immunity and protect the animals from lethal challenge with SIV, the cognate MV/HIV-1 recombinants will be constructed with the aim to generate live vaccines which in conjunction with other vectors expressing HIV-1 antigens might provide complete protection against HIV infection.
2. Materials and methods
2.1. Plasmid constructions All cloning procedures were carried out basically as described by Sambrook et al. [11]. PCR amplifications were performed using the proof-reading pfu DNA polymerase (Stratagene) and primers bearing a 3%-terminal phosphorothioate bond instead of a phosphodiester bond. All constructs are derivatives of plasmid p(+) MV which specifies antigenomes of the MV tag Edmonston B, precisely initiated by T7 RNA polymerase and precisely terminated by the hepatitis d ribozyme
Fig. 1. Construction of recombinant MV/MuV and MV/SIV plasmids. p( +)MV-HN2-MuV, p(+ )MV-F2-MuV, p(+)MV-env2-SIV, p( +)MVenv3-SIV, p( + )MV-pol2-SIV and p( + )MV-gag3-SIV were constructed based on p( +)MPrGFPV, and p(+)MHrGFPV [4]. For SIV inserts, PCR products were first cloned in a T-vector (see Section 2), then transferred into the MV genome contest via BssH II and SnaB I. The rGFP reading frames from the acceptor plasmids were replaced by those of SIV as indicated. For MuV inserts encoding HN and F, PCR products were digested with BssH II and Aat II, and used directly to replace the rGFP insert in the acceptor plasmid.
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[12]. From this progenitor (Fig. 1, top) three vector plasmids had been derived bearing additional transcription units which contained suitable unique restriction sites for easy exchange of open reading frames (ORFs). One vector plasmid contains the additional transcription unit upstream of the N gene (position 1), a second plasmid, downstream of the P gene (p(+ )MPrGFPV, position 2; Fig. 1, second panel), and the third plasmid, downstream of the H gene (p(+ )MHrGFPV, position 3; Fig. 1, third panel); in these and all subsequent constructs care was taken to maintain the ‘rule of six’, keeping the total number of antigenomic nucleotides as a multiple of six [13]. The two plasmids containing the additional transcription units in position 2 and 3, respectively, were used for the cloning of MuV HN or F and of SIV env pol or gag ORFs (Fig. 1). The HN and F ORFs were amplified from the plasmids p(+ )GC4HN and p( + ) GC19F (a gift from Dr M.G. Cusi, Siena, Italy. For HN, two primers 5%-CCGACGCGTTCCATGGAGCCCTCCAAACTCTTC-3% and 5%-GGCGACGTCCCTTATCAAGTGATGGTCAATC-3%, and for F, the primers 5%-CCGACGCGTCATGGCGGCTTTTTTAGTTACTTGCTTAAG-3% and 5%-GGCGACGTCCTTAGTACCTAATGAGATCATC-3% were used. The PCR products were flanked by the two restriction sites Mlu I (from the 5% end) and Aat II (from the 3% end), both underlined. p(+)MPrGFPV was cut by these restriction enzymes and the PCR fragment containing HN or F genes replaced that of rGFP. The resulting antigenomic plasmids were verified by sequencing and designated p(+)MV-HN2-Mu and p( + )MV-F2-Mu, respectively. The SIV gag and pol genes were amplified by PCR from the p239SpSp5 plasmid, and env from the plasmid p239SpE3 (gifts of R. Andino, San Francisco, CA). Both plasmids contain genomic regions of the SIV strain 239-1. The forward PCR primer for the gag gene was 5%-CGTACGTAGATGGGCGTGAGAAACTCCGTC-3%, and the reverse primer 5%-TTGGCGCGCTGACTACTGGTCTCCTCCAAAGAGAG3%. The pol primers were 5%-GTACGTAATGGTGTTGGAATTGTGGGAAAG-3% and 5%-TTGCGCGCTATGAGGCTATGCCACCTCTCT-3%. The env primers were, 5%-CGTACGTACACAAGTA AGTATGGGATGTC-3% and 5%-TGCGCGCTATCACAAGAGAGTGAGCTCAAG-3%. All forward primers contained a SnaB I site and all reverse primers a BssH II site both underlined. The gag, pol and env PCR products were terminally adenylated by incubation with 5 U Taq DNA polymerase in Taq polymerase reaction buffer, 2.5 mM MgCl and 0.2 mM dATP (Promega) for 30 min at 72°C. A-tailed PCR fragments were subcloned into the pGEM-T vector (Promega), and insert genotypes were confirmed by sequence analysis. The SnaB I/BssH II
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restriction sites flanking the inserted genes were then used to clone SIV gag, pol and env sequences directly into the respective antigenomic MV vectors. The resulting plasmids were designated p(+ )MV-gag3-SIV, p(+) MV-pol2-SIV, p(+ )MV-env2-SIV, and p(+)MVenv3-SIV, respectively; the numbers refer to the closing position within the MV vector. Rescue of the recombinant viruses, infectivity assays and growth curves procedures were carried out as described previously [5,14].
2.2. Assays for protein expression and for transgene stability Rescued viruses were serially passaged 10 times in Vero cells at an MOI of about 0.01; 1% of the supernatant of each culture was used to infect the subsequent Vero cell layer. To test transgene expression and stability, passage 1, passage 5 and passage 10 viruses were used for further characterization of the expression of MuV and SIV antigens by immunofluorescence and biochemical characterization (radiolabeling followed by immunoprecipitation and SDS-PAGE, or by Western blot analysis), essentially as described previously [5,15].
3. Results
3.1. Antigenomic plasmid constructions and rescue of recombinant MV/MuV and MV/SIV 6iruses The ORFs encoding SIV and MuV proteins were amplified, cloned into p(+)MPrGFPV or p( +) MHrGFPV, and rMVs were rescued with the helper cell line 293-3-46 (see Section 2, and Fig. 1). ORFs encoding the red-shifted green fluorescent protein (rGFP) had been inserted previously as separate MVspecific transcription units at three different loci in the MV genome, upstream of N gene (position 1), between the P and the M gene (position 2), and between the H and the L gene (position 3). The transgene expression levels depend on their site of insertion in the genome. The relative expression from transcription units added at positions 1, 2 and 3 amounts to an mRNA molar ratio of about 10:3:1 [4]. It should be mentioned here that the replication efficiency of subgenomic MV replicons strongly depends on the so-called ‘rule of six’, a notion which has been confirmed later to hold true for the entire genome as well [16]. This rule, which had been discovered by Roux and collaborators for defective interfering particle RNA of Sendai virus (SeV), states that efficient replication of subgenomic SeV takes place only when the nucleotide number of the replicating entity is a multiple of six [13]. Therefore, care was taken to construct all antigenomic MV plasmids to conform to this rule.
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Fig. 2. Expression of MuV and SIV proteins in recombinant MV. Vero cells were infected with rMVs at an MOI of 0.1 for 48 h in the case of MV/SIV, and till 60 h for MV/MuV recombinant viruses. (A) Expression of MuV F and HN was determined by immunoprecipitation of labeled proteins. Cells were pulse labeled for 3 h, lysed clarified, and monoclonal anti-Mu-HN (lane 1), F (lane 3) or anti-MV H (lanes 2 and 4) antibodies were used. Immunoprecipitates were washed and proteins analyzed by 12% SDS-PAGE with detection using a phosphorimager. (B) To determine expression of SIV proteins env, pol and gag, infected cells were lysed and the clarified lysates were resolved on 10% SDS-PAGE, blotted onto Immobilon membranes (Millipore) and probed with a monkey polyclonal anti SIV antibody subsequently stained with a goat anti-monkey-HRP secondary antibody. Proteins were then detected using an ECL detection kit (Amersham Pharmacia Biotech). Lane 1: reactivity of SIV antibody with an MV lysate; lane 2: rMV containing env in position 2; lane 3: rMV containing env in position 3; lane 4: rMV containing pol in position 2; lane 5: rMV containing gag in position 3.
The efficiency of virus rescue was roughly similar for all recombinant antigenomic plasmids; however, the onset of visible rescue as monitored by syncytia formation was delayed to various degrees in comparison to the standard p(+)MV. Whereas standard MV normally rescues at days 4 or 5 after transfection, the onset of syncytia formation of the rMVs ranged from 7 to 10 days. Two syncytia were picked from each individual transfection for preparation of separate virus stocks.
3.2. Expression of MuV and SIV proteins The rescued recombinant viruses, MV-F2-Mu, MVHN2-Mu, MV-env2-SIV, MV-env3-SIV, MV-gag3-SIV and MV-pol2-SIV, were propagated in Vero cells. Two diverse approaches were used to monitor transgene expression: immunofluorescence (not shown) and biochemical characterisation (Fig. 2). The expression of MuV-HN and -F, and SIV-env and -pol by the rMVs was confirmed by immunofluorescence. All observed syncytia elicited by rMVs were found to express the inserted transgenes together with MV genes as expected; in contrast, standard MVtriggered syncytia expressed only MV genes (not shown). SIV-gag expression could not be detected by
this assay. Therefore, to ascertain the expression of MuV HN and F as well as SIV env, gag and pol proteins expressed by the rMVs biochemical analysis was performed. Cells were infected with the corresponding rMV with an MOI of 0.1, and cells were either directly lysed and immunoblotted (for MV/SIV), or were pulse labeled for 3 h, and then lysed for immunoprecipitation with the specific antibodies (for MV/MuV). The results are shown in Fig. 2. Infection with MV/MuV recombinants showed extensive expression of MuV-HN protein (Fig. 2A, lane 1). Compared to the level of MV-H expression, MuV-HN was about two- to threefold higher (lanes 1 and 2). Compared to MV-H in the same experiment, Mu-F was also detected but in significantly lower amounts (lanes 3 and 4). As a negative control, lysates from the Standard MV (MVtag) infected cells were probed with anti-Mu-HN and F antibodies, which showed no reactivity (lane 5). As expected, the MV-env2-SIV, MV-env3-SIV viruses expressed the env precursor, gp160, and the cleaved gp120 proteins (Fig. 3B, lanes 2 and 3). In the case of MV-pol2-SIV, the pol precursor, p165 protein was detected (lane 4). With the recombinant bearing env at position 3, lower expression of gp160 was observed (lane 3) which reflects fact that mRNA and
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concomitantly protein-expression from the MV genome is regulated by a transcription gradient. As a negative control, lysates from the MV-tag infected cells were probed with anti-SIV antibody. The results show no reactivity of the SIV antibody with any of the MV proteins (lane 1). Taken together, these results suggest that all SIV proteins (env, gag and pol) and Mu-HN are efficiently expressed by the respective rMVs; Mu-F on the other hand was expressed at lower levels, which might be due to the instability of the cognate transgene (see below).
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3.3. Stability of transgene protein expression by recombinant MVs To determine whether the expression of the cloned MuV and SIV genes is stably maintained, the rescued rMVs were passaged in parallel with the standard MVtag for 10 serial transfers in Vero cells. Expression of the recombinant proteins was analysed at passages 1, 5 and 10, in the case of MV/SIV recombinants by immunofluorescence assays (Fig. 3A), and for MV/MuV recombinants by SDS-PAGE (Fig. 3B).
Fig. 3. Stability of transgene expression in rMVs. After serial passages on Vero cells, protein expression was determined either by immunofluorescence in the case of MV/SIV recombinant viruses (A), or by immunoprecipitation for MV/MuV recombinant viruses (B). (A) SIV pol and env were detected by polyclonal anti-SIV antibody coupled to FITC (green). In the same syncytia, MV-anti-F antibody coupled to Cy3 (red) was used as an internal control. Overlap images were reconstituted showing coexpression of the recombinant proteins and MV marker protein. Typical syncytia of passage 10 are shown. Bars, 10 mm. (B) Stability of MuV HN and F protein expression in serial passages. Vero cells were infected with viruses from passage 1, 5 or 10, labeled and proteins were immunoprecipitated by the specific antibody. MV-H was immunoprecipitated from the corresponding lysates as an internal control.
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Fig. 4. Growth kinetics of recombinant MV/SIV and MV/MuV viruses. Vero cells on multi-well 36-mm dishes were infected with MVtag, MV-HN2-Mu, MV-F2-Mu, MV-env2-SIV, MV-env3-SIV and MV-pol2-SIV at an MOI of 0.01. To increase the sensitivity for differences in growth kinetics the cultures were kept at 32.5°C. At each time point, media (cell-free) and cells were collected separately. Virus titers were determined by plaque assay.
In cells infected with MV/SIV recombinant viruses, more than 50 syncytia were analyzed for each monitored passage. All counted syncytia were positive for SIV proteins after 10 passages. Typical expression is shown in Fig. 3A, which shows coexpression of SIV and MV proteins. For standard MV used as a negative control, only F expression was detected (Fig. 3A). These results indicate clearly that SIV genes in rMV are extremely stable even after a virus amplification by a factor of 1020. In MV/MuV recombinant viruses, Mu-HN protein was also rather stably expressed (Fig. 3B). In contrast, F expression was not as stable, expression was lost gradually from passages 1 to 10 (Fig. 3B). Most likely, Mu-F strongly interferes with the similar cognate MVF in progeny formation. In this view, spontaneously arising mutants in which the MuF reading frame is interrupted would have a strong competitive advantage, and would overgrow the intact recombinant. In fact, it was observed that MV-F2-MuV propagated very slowly in the initial passages, whereas in late passages its growth kinetics became similar to that of the standard MV (results not shown).
3.4. Heterologous genes affect the growth kinetics of rMVs to 6arious degrees The growth of all rescued recombinant viruses was slower then standard MV. To determine the growth kinetics of these viruses, Vero cells in multi-well plates were infected at an MOI of 0.1 and incubated at 32.5°C for various times (24, 48, 60, 72, 84 and 96 h). At each time point, media and cells were separately collected, and frozen until the end of the observation period. Titers of cell-free and cell-associated viruses were then determined for each time point. The results are shown in Fig. 4. Compared to standard MV, the growth kinetics of MV-pol2-SIV were only slightly affected (a delay of about 12 h to reach the peak titer), whereas a delay of about 24 h was observed for both MV-env2SIV and MV-env3-SIV viruses. Comparing the peak titers of cell-free viruses, standard MV reached 2×105 pfu/ml, MV-env2-SIV 4.4× 104, MV-env3-SIV 3×104, and MV-pol2-SIV 1.2× 105. The peak titers of cell-associated viruses were 1× 106 pfu/ml for the standard MV, 4.5 ×105 for MV-env2-SIV, 4× 105 for MV-env3SIV, and 6 ×105 for MV-pol2-SIV. MV-gag3-SIV grew similarly to MV-pol2-SIV (not shown).
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For recombinant MV/MuV viruses the growth kinetics were even slower. Compared to the standard MV, both MV-HN2-Mu and MV-F2-Mu were about 48 – 60 h slower, but the peak titers of cell-free and cell-associated viruses were comparable to those of MV/SIV recombinant viruses (Fig. 4). These results indicate that enriching the MV genome by additional genes encoding viral proteins affects the viral propagation speed to various degrees. Whereas SIV proteins had minor effects on the growth kinetics, MuV proteins substantially retarded the propagation of the respective viruses. Thus, the MV/MuV recombinants are not suitable as candidate vaccines. More generally, it may be concluded that for vaccination purposes it is not suitable to construct recombinants based on a paramyxovirus expressing additional components of another paramyxovirus. However, the construction of chimera in which components of one paramyxovirus are substituted by other paramyxovirus components [5] might be useful for particular purposes.
4. Discussion We have genetically engineered measles viruses for possible use as efficacious multivalent live-vaccine candidates. The results described here show that the non-segmented, negative-strand measles virus genome has the potential not only to stably express additional short or large heterologous transcripts of cytoplasmically expressed marker genes like CAT, GFP, and b-gal [4,17] or small virus-derived genes such as those encoding HBV c and s antigens [7], but also complete ORFs of viral genes exceeding 3 kb. Many viral systems have limitations in their ability to accept and stably maintain the expression of long transgenes. The successful rescue and subsequent spread of measles viruses expressing large transgenes over many rounds of replication suggest the utility of products as vaccine vectors, given the high efficacy and safety of attenuated MVs as vaccines. However, a limitation to this approach has been delineated here, specifically the interference of transgene from a closely related virus such as MuV, which was not observed in the case of unrelated viruses, specifically HBV as shown earlier [7] and SIV as shown here. Expression of the MuV virus surface glycoproteins in the presence of the normal complement of MV surface glycoproteins negatively affects the propagation kinetics of these particular rMVs, and results in a negative selection pressure. This can eventually lead to the interruption of such reading frames by spontaneous mutations, thus almost restoring the growth kinetics to that of standard MV, despite the maintenance of the large insert altered only by point mutations. Importantly, SIV protein expression was more stable, and affected the propagation of the cognate rMVs only slightly.
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Thus, the construction of rMVs carrying transgenes of other pathogens appears now promising. In particular, if macaques inoculated with the MV/SIV recombinants shown in this paper and with rMVs expressing nef and tat regulatory proteins (F. Tangy, Institut Pasteur, Paris, unpublished results) show strong immune responses and protection against lethal challenge with SIV, the construction and testing of analogous MV/HIV recombinants is warranted. Although no universal consensus exists on what components will be needed in a vaccine that is able to induce protective immunity against HIV-1 infection, such MV/HIV recombinants appear at least as appropriate as candidates using other vector systems. In fact, it might be necessary to utilize different vectors in successive boost vaccinations to obtain complete protection. The current view is that both the humoral and the cellular arms of the human immune system should be efficiently stimulated [4], which should be attainable using rMVs expressing a set of entire env, gag, pol, nef and tat proteins. The Edmonston B strain of measles is an approved, safe and efficacious vaccine that induces both long lasting, humoral and cellular immunity. The reason for this high efficacy and safety, even in comparison with the highly successful live polio vaccine is not understood so far, and might at least in part be due to the cell tropism, efficiently targeting antigen presenting cells such as macrophages and dendritic cells [2,18]. Thus, rMVs expressing antigens of very serious pathogens against which no vaccines exist so far, such as HIV, plasmodia eliciting Malaria and hepatitis C virus, might be particularly suitable to combat the corresponding diseases in developing countries. One negative aspect for the use of rMVs is the fact that in almost all individuals of the adult population there is pre-existing immunity against MV, due to either a history with acute measles or a measles vaccination in early childhood. However, the potential benefits of immunizing children early in life with a cocktail of rMVs expressing antigens of all major diseases rather than with a standard MV vaccine, as currently enforced world-wide by the WHO measles eradication campaign must not be ignored. In addition, it has been shown that school children, when reimmunized with MV, mount important recall immune responses [18], contrary to the lack of anti-MV immune induction exhibited by small infants containing a threshold level of maternal anti-MV antibodies [19]. The response of older children to repeat MV vaccinations in comparison to the lack of immunization response in small infants may be due to the immature immune system of the latter. Thus, it is conceivable that also in adults vaccination with rMV cocktails will induce at least partial immunity against the pathogens from which the transgenes inserted into the MV vector were derived. The
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particularly serious morbidity usually monitored in first infections by a particular pathogen might thereby be circumvented. Nevertheless, reasons for caution exist. First, measles induces strong immunosuppression, as does the attenuated standard MV vaccine albeit to a minor extent. The even more attenuated rMVs will probably also exhibit at least some of this property, which in the case of individuals having contracted HIV before could constitute a contraindication for the use of any live MV vaccine. Second, rMVs expressing envelope glycoproteins of other viruses might acquire a cell tropism differing from that of the standard live MV vaccines. This might be the case even if the foreign glycoprotein is not incorporated into MV particles, since MV spread within a host seems to occur primarily by cell/cell fusion [15,20,21]. Only experiments with primates will reveal whether these dangers are indeed serious in reality. In any event, rMVs could be constructed in which the fusion protein cannot be cleaved in the host, thus allowing in principle only one round of replication [22]. A drawback of such propagation-deficient rMVs would be the much larger doses required for each immunization, thus increasing the production cost and therefore the potential for the use of such vaccines in developing countries.
Acknowledgements Active collaboration with F. Tangy, Institut Pasteur, Paris, France, including exchange of results and materials is gratefully acknowledged. Plasmids bearing MuV and SIV genes were provided by Dr M.G. Cusi, Siena, Italy and by Dr R. Andino, San Francisco, CA, respectively. We thank Dr R. Glu¨ck of the Swiss Serum and Vaccine Institute (Berna) for his constant interest, discussions and support. This work was supported by grants from the Swiss National Science Foundation, 31.45900-95, the Commission for Technology and Innovation, 3786.1, and the NIH grant A146007-01.
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Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses Z. Wang, L. Hangartner, T.I. Cornu, L.R. Martin, A. Zuniga, M.A. Billeter *, H.Y. Naim Institute of Molecular Biology, Uni6ersity of Zurich-Irchel, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Abstract We have genetically engineered a panel of recombinant measles viruses (rMVs) that express from various positions within the MV genome either the HN or F surface glycoproteins of mumps virus (MuV) or the env, gag or pol proteins from simian immunodeficiency virus (SIV). All rMVs were rescued from the respective antigenomic plasmid constructs; progeny viruses replicated comparably to the progenitor Edmonston B MV, but showed slight propagation retardation, which was dependent on the size and nature of the expressed proteins and on the genomic position of the inserts. All transgenes except that encoding mumps F glycoprotein were faithfully maintained and expressed even after virus amplification by 1020. Our results suggest possible applications of rMVs as live-attenuated, multivalent vaccines against retroviruses such as SIV and HIV as well as other pathogens more distantly related to MV than MuV. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Attenuated measles viruses; Live vaccines; Expression vectors
1. Introduction Measles virus (MV), an enveloped virus with a nonsegmented, tightly encapsidated negative-strand RNA genome, belongs to the family Paramyxo6iride, genus Morbilli6irus [1,2]. MV cDNA plasmids have been constructed to produce precisely initiated and terminated MV antigenomes related to the Edmonston B vaccine strains, and a helper-cell-based system has been established which allows the rescue of MV from such MV antigenomic plasmids [3]. This allows the construction of genetically altered MV and of recombinant derivatives of MV (rMVs) expressing proteins from inserted transgenes [4,5]. The most complex rMV generated so far, exceeding in length the standard MV by more than 5000 nucleotides, contains the coding regions for green fluorescent protein (GFP), beta-galactosidase (b-gal) and chloramphenicol acetyl transferase (CAT), engineered into three positions of the MV antigenome; the inserted coding regions are expressed as independent transcription units [4]. These transgenes were expressed * Corresponding author. Tel.: +41-1-6353123; fax: +41-16356864. E-mail address: [email protected] (M.A. Billeter).
at levels, according to their genomic position, and were stably maintained over many viral generations. This is rather surprising for an RNA virus-based vector, since all RNA polymerases are error-prone, and positivestrand RNA viruses are notorious for spontaneous deletion of inserted genetic material, presumably by nonhomologous copy-choice recombination events. Recombinant MVs expressing biologically relevant proteins such as IL-12 or small proteins derived from other pathogens, including the small surface protein and the core protein of hepatitis B virus (HBV), have shown only minor propagation delays and little impairment of end titers [6,7]. In addition, the complete replacement of the MV envelope proteins F and H by the single envelope protein G of the distantly related vesicular stomatitis virus (VSV) generated stable chimeric viruses (MV/VSV) that gained the tropism of VSV [5]. The ability to construct new recombinant and chimeric MVs opens the prospect to develop new vaccines based on MV. The limited immunization experiments carried out so far with rMVs have been encouraging. The MV expressing HBV antigens induced vigorous humoral immune responses in transgenic mice, which have been made susceptible to MV
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replication by insertion of a YAC encompassing the gene encoding the MV receptor CD46 and elimination of the interferon type 1 receptor [8,9]. Squirrel monkeys Saimiri inoculated with b-gal-expressing MV generated both strong humoral and cellular immune responses against b-gal, (F. Tangy, pers. commun.), although b-gal is inherently not a potent immunogen. Interferon defective mice were protected against lethal doses of VSV by immunization with the MV-VSV chimera [10]. In comparison with other viral vector systems in use for the delivery of foreign proteins and for immunization purposes, the MV Edmonston vaccine strain-based vector system has decisive advantages. The Edmonston B strain has a long history as a safe and efficacious vaccine in human subjects, inducing life-long protection against measles usually by a single application; furthermore, the production cost of vaccination doses is very low. It may be expected that rMVs can induce longlasting immunity both against MV and the pathogens from which the inserted transgenes are derived. In the present study, we investigated the feasibility to generate rMVs expressing transgenes derived either from the mumps virus (MuV), which is relatively closely related to MV, or simian immunodeficiency virus (SIV), to see whether the expression of large viral components strongly interferes with the propagation of the vector. In contrast to one MV/MuV recombinant,
all MV/SIV recombinants turned out to stably maintain the expression of the inserted reading frames. The latter recombinants are of particular interest; if immunization of macaques with a mixture of these and other recombinants expressing SIV tat and nef (F. Tangy, unpublished results) will induce both strong humoral and cell-mediated immunity and protect the animals from lethal challenge with SIV, the cognate MV/HIV-1 recombinants will be constructed with the aim to generate live vaccines which in conjunction with other vectors expressing HIV-1 antigens might provide complete protection against HIV infection.
2. Materials and methods
2.1. Plasmid constructions All cloning procedures were carried out basically as described by Sambrook et al. [11]. PCR amplifications were performed using the proof-reading pfu DNA polymerase (Stratagene) and primers bearing a 3%-terminal phosphorothioate bond instead of a phosphodiester bond. All constructs are derivatives of plasmid p(+) MV which specifies antigenomes of the MV tag Edmonston B, precisely initiated by T7 RNA polymerase and precisely terminated by the hepatitis d ribozyme
Fig. 1. Construction of recombinant MV/MuV and MV/SIV plasmids. p( +)MV-HN2-MuV, p(+ )MV-F2-MuV, p(+)MV-env2-SIV, p( +)MVenv3-SIV, p( + )MV-pol2-SIV and p( + )MV-gag3-SIV were constructed based on p( +)MPrGFPV, and p(+)MHrGFPV [4]. For SIV inserts, PCR products were first cloned in a T-vector (see Section 2), then transferred into the MV genome contest via BssH II and SnaB I. The rGFP reading frames from the acceptor plasmids were replaced by those of SIV as indicated. For MuV inserts encoding HN and F, PCR products were digested with BssH II and Aat II, and used directly to replace the rGFP insert in the acceptor plasmid.
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[12]. From this progenitor (Fig. 1, top) three vector plasmids had been derived bearing additional transcription units which contained suitable unique restriction sites for easy exchange of open reading frames (ORFs). One vector plasmid contains the additional transcription unit upstream of the N gene (position 1), a second plasmid, downstream of the P gene (p(+ )MPrGFPV, position 2; Fig. 1, second panel), and the third plasmid, downstream of the H gene (p(+ )MHrGFPV, position 3; Fig. 1, third panel); in these and all subsequent constructs care was taken to maintain the ‘rule of six’, keeping the total number of antigenomic nucleotides as a multiple of six [13]. The two plasmids containing the additional transcription units in position 2 and 3, respectively, were used for the cloning of MuV HN or F and of SIV env pol or gag ORFs (Fig. 1). The HN and F ORFs were amplified from the plasmids p(+ )GC4HN and p( + ) GC19F (a gift from Dr M.G. Cusi, Siena, Italy. For HN, two primers 5%-CCGACGCGTTCCATGGAGCCCTCCAAACTCTTC-3% and 5%-GGCGACGTCCCTTATCAAGTGATGGTCAATC-3%, and for F, the primers 5%-CCGACGCGTCATGGCGGCTTTTTTAGTTACTTGCTTAAG-3% and 5%-GGCGACGTCCTTAGTACCTAATGAGATCATC-3% were used. The PCR products were flanked by the two restriction sites Mlu I (from the 5% end) and Aat II (from the 3% end), both underlined. p(+)MPrGFPV was cut by these restriction enzymes and the PCR fragment containing HN or F genes replaced that of rGFP. The resulting antigenomic plasmids were verified by sequencing and designated p(+)MV-HN2-Mu and p( + )MV-F2-Mu, respectively. The SIV gag and pol genes were amplified by PCR from the p239SpSp5 plasmid, and env from the plasmid p239SpE3 (gifts of R. Andino, San Francisco, CA). Both plasmids contain genomic regions of the SIV strain 239-1. The forward PCR primer for the gag gene was 5%-CGTACGTAGATGGGCGTGAGAAACTCCGTC-3%, and the reverse primer 5%-TTGGCGCGCTGACTACTGGTCTCCTCCAAAGAGAG3%. The pol primers were 5%-GTACGTAATGGTGTTGGAATTGTGGGAAAG-3% and 5%-TTGCGCGCTATGAGGCTATGCCACCTCTCT-3%. The env primers were, 5%-CGTACGTACACAAGTA AGTATGGGATGTC-3% and 5%-TGCGCGCTATCACAAGAGAGTGAGCTCAAG-3%. All forward primers contained a SnaB I site and all reverse primers a BssH II site both underlined. The gag, pol and env PCR products were terminally adenylated by incubation with 5 U Taq DNA polymerase in Taq polymerase reaction buffer, 2.5 mM MgCl and 0.2 mM dATP (Promega) for 30 min at 72°C. A-tailed PCR fragments were subcloned into the pGEM-T vector (Promega), and insert genotypes were confirmed by sequence analysis. The SnaB I/BssH II
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restriction sites flanking the inserted genes were then used to clone SIV gag, pol and env sequences directly into the respective antigenomic MV vectors. The resulting plasmids were designated p(+ )MV-gag3-SIV, p(+) MV-pol2-SIV, p(+ )MV-env2-SIV, and p(+)MVenv3-SIV, respectively; the numbers refer to the closing position within the MV vector. Rescue of the recombinant viruses, infectivity assays and growth curves procedures were carried out as described previously [5,14].
2.2. Assays for protein expression and for transgene stability Rescued viruses were serially passaged 10 times in Vero cells at an MOI of about 0.01; 1% of the supernatant of each culture was used to infect the subsequent Vero cell layer. To test transgene expression and stability, passage 1, passage 5 and passage 10 viruses were used for further characterization of the expression of MuV and SIV antigens by immunofluorescence and biochemical characterization (radiolabeling followed by immunoprecipitation and SDS-PAGE, or by Western blot analysis), essentially as described previously [5,15].
3. Results
3.1. Antigenomic plasmid constructions and rescue of recombinant MV/MuV and MV/SIV 6iruses The ORFs encoding SIV and MuV proteins were amplified, cloned into p(+)MPrGFPV or p( +) MHrGFPV, and rMVs were rescued with the helper cell line 293-3-46 (see Section 2, and Fig. 1). ORFs encoding the red-shifted green fluorescent protein (rGFP) had been inserted previously as separate MVspecific transcription units at three different loci in the MV genome, upstream of N gene (position 1), between the P and the M gene (position 2), and between the H and the L gene (position 3). The transgene expression levels depend on their site of insertion in the genome. The relative expression from transcription units added at positions 1, 2 and 3 amounts to an mRNA molar ratio of about 10:3:1 [4]. It should be mentioned here that the replication efficiency of subgenomic MV replicons strongly depends on the so-called ‘rule of six’, a notion which has been confirmed later to hold true for the entire genome as well [16]. This rule, which had been discovered by Roux and collaborators for defective interfering particle RNA of Sendai virus (SeV), states that efficient replication of subgenomic SeV takes place only when the nucleotide number of the replicating entity is a multiple of six [13]. Therefore, care was taken to construct all antigenomic MV plasmids to conform to this rule.
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Fig. 2. Expression of MuV and SIV proteins in recombinant MV. Vero cells were infected with rMVs at an MOI of 0.1 for 48 h in the case of MV/SIV, and till 60 h for MV/MuV recombinant viruses. (A) Expression of MuV F and HN was determined by immunoprecipitation of labeled proteins. Cells were pulse labeled for 3 h, lysed clarified, and monoclonal anti-Mu-HN (lane 1), F (lane 3) or anti-MV H (lanes 2 and 4) antibodies were used. Immunoprecipitates were washed and proteins analyzed by 12% SDS-PAGE with detection using a phosphorimager. (B) To determine expression of SIV proteins env, pol and gag, infected cells were lysed and the clarified lysates were resolved on 10% SDS-PAGE, blotted onto Immobilon membranes (Millipore) and probed with a monkey polyclonal anti SIV antibody subsequently stained with a goat anti-monkey-HRP secondary antibody. Proteins were then detected using an ECL detection kit (Amersham Pharmacia Biotech). Lane 1: reactivity of SIV antibody with an MV lysate; lane 2: rMV containing env in position 2; lane 3: rMV containing env in position 3; lane 4: rMV containing pol in position 2; lane 5: rMV containing gag in position 3.
The efficiency of virus rescue was roughly similar for all recombinant antigenomic plasmids; however, the onset of visible rescue as monitored by syncytia formation was delayed to various degrees in comparison to the standard p(+)MV. Whereas standard MV normally rescues at days 4 or 5 after transfection, the onset of syncytia formation of the rMVs ranged from 7 to 10 days. Two syncytia were picked from each individual transfection for preparation of separate virus stocks.
3.2. Expression of MuV and SIV proteins The rescued recombinant viruses, MV-F2-Mu, MVHN2-Mu, MV-env2-SIV, MV-env3-SIV, MV-gag3-SIV and MV-pol2-SIV, were propagated in Vero cells. Two diverse approaches were used to monitor transgene expression: immunofluorescence (not shown) and biochemical characterisation (Fig. 2). The expression of MuV-HN and -F, and SIV-env and -pol by the rMVs was confirmed by immunofluorescence. All observed syncytia elicited by rMVs were found to express the inserted transgenes together with MV genes as expected; in contrast, standard MVtriggered syncytia expressed only MV genes (not shown). SIV-gag expression could not be detected by
this assay. Therefore, to ascertain the expression of MuV HN and F as well as SIV env, gag and pol proteins expressed by the rMVs biochemical analysis was performed. Cells were infected with the corresponding rMV with an MOI of 0.1, and cells were either directly lysed and immunoblotted (for MV/SIV), or were pulse labeled for 3 h, and then lysed for immunoprecipitation with the specific antibodies (for MV/MuV). The results are shown in Fig. 2. Infection with MV/MuV recombinants showed extensive expression of MuV-HN protein (Fig. 2A, lane 1). Compared to the level of MV-H expression, MuV-HN was about two- to threefold higher (lanes 1 and 2). Compared to MV-H in the same experiment, Mu-F was also detected but in significantly lower amounts (lanes 3 and 4). As a negative control, lysates from the Standard MV (MVtag) infected cells were probed with anti-Mu-HN and F antibodies, which showed no reactivity (lane 5). As expected, the MV-env2-SIV, MV-env3-SIV viruses expressed the env precursor, gp160, and the cleaved gp120 proteins (Fig. 3B, lanes 2 and 3). In the case of MV-pol2-SIV, the pol precursor, p165 protein was detected (lane 4). With the recombinant bearing env at position 3, lower expression of gp160 was observed (lane 3) which reflects fact that mRNA and
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concomitantly protein-expression from the MV genome is regulated by a transcription gradient. As a negative control, lysates from the MV-tag infected cells were probed with anti-SIV antibody. The results show no reactivity of the SIV antibody with any of the MV proteins (lane 1). Taken together, these results suggest that all SIV proteins (env, gag and pol) and Mu-HN are efficiently expressed by the respective rMVs; Mu-F on the other hand was expressed at lower levels, which might be due to the instability of the cognate transgene (see below).
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3.3. Stability of transgene protein expression by recombinant MVs To determine whether the expression of the cloned MuV and SIV genes is stably maintained, the rescued rMVs were passaged in parallel with the standard MVtag for 10 serial transfers in Vero cells. Expression of the recombinant proteins was analysed at passages 1, 5 and 10, in the case of MV/SIV recombinants by immunofluorescence assays (Fig. 3A), and for MV/MuV recombinants by SDS-PAGE (Fig. 3B).
Fig. 3. Stability of transgene expression in rMVs. After serial passages on Vero cells, protein expression was determined either by immunofluorescence in the case of MV/SIV recombinant viruses (A), or by immunoprecipitation for MV/MuV recombinant viruses (B). (A) SIV pol and env were detected by polyclonal anti-SIV antibody coupled to FITC (green). In the same syncytia, MV-anti-F antibody coupled to Cy3 (red) was used as an internal control. Overlap images were reconstituted showing coexpression of the recombinant proteins and MV marker protein. Typical syncytia of passage 10 are shown. Bars, 10 mm. (B) Stability of MuV HN and F protein expression in serial passages. Vero cells were infected with viruses from passage 1, 5 or 10, labeled and proteins were immunoprecipitated by the specific antibody. MV-H was immunoprecipitated from the corresponding lysates as an internal control.
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Fig. 4. Growth kinetics of recombinant MV/SIV and MV/MuV viruses. Vero cells on multi-well 36-mm dishes were infected with MVtag, MV-HN2-Mu, MV-F2-Mu, MV-env2-SIV, MV-env3-SIV and MV-pol2-SIV at an MOI of 0.01. To increase the sensitivity for differences in growth kinetics the cultures were kept at 32.5°C. At each time point, media (cell-free) and cells were collected separately. Virus titers were determined by plaque assay.
In cells infected with MV/SIV recombinant viruses, more than 50 syncytia were analyzed for each monitored passage. All counted syncytia were positive for SIV proteins after 10 passages. Typical expression is shown in Fig. 3A, which shows coexpression of SIV and MV proteins. For standard MV used as a negative control, only F expression was detected (Fig. 3A). These results indicate clearly that SIV genes in rMV are extremely stable even after a virus amplification by a factor of 1020. In MV/MuV recombinant viruses, Mu-HN protein was also rather stably expressed (Fig. 3B). In contrast, F expression was not as stable, expression was lost gradually from passages 1 to 10 (Fig. 3B). Most likely, Mu-F strongly interferes with the similar cognate MVF in progeny formation. In this view, spontaneously arising mutants in which the MuF reading frame is interrupted would have a strong competitive advantage, and would overgrow the intact recombinant. In fact, it was observed that MV-F2-MuV propagated very slowly in the initial passages, whereas in late passages its growth kinetics became similar to that of the standard MV (results not shown).
3.4. Heterologous genes affect the growth kinetics of rMVs to 6arious degrees The growth of all rescued recombinant viruses was slower then standard MV. To determine the growth kinetics of these viruses, Vero cells in multi-well plates were infected at an MOI of 0.1 and incubated at 32.5°C for various times (24, 48, 60, 72, 84 and 96 h). At each time point, media and cells were separately collected, and frozen until the end of the observation period. Titers of cell-free and cell-associated viruses were then determined for each time point. The results are shown in Fig. 4. Compared to standard MV, the growth kinetics of MV-pol2-SIV were only slightly affected (a delay of about 12 h to reach the peak titer), whereas a delay of about 24 h was observed for both MV-env2SIV and MV-env3-SIV viruses. Comparing the peak titers of cell-free viruses, standard MV reached 2×105 pfu/ml, MV-env2-SIV 4.4× 104, MV-env3-SIV 3×104, and MV-pol2-SIV 1.2× 105. The peak titers of cell-associated viruses were 1× 106 pfu/ml for the standard MV, 4.5 ×105 for MV-env2-SIV, 4× 105 for MV-env3SIV, and 6 ×105 for MV-pol2-SIV. MV-gag3-SIV grew similarly to MV-pol2-SIV (not shown).
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For recombinant MV/MuV viruses the growth kinetics were even slower. Compared to the standard MV, both MV-HN2-Mu and MV-F2-Mu were about 48 – 60 h slower, but the peak titers of cell-free and cell-associated viruses were comparable to those of MV/SIV recombinant viruses (Fig. 4). These results indicate that enriching the MV genome by additional genes encoding viral proteins affects the viral propagation speed to various degrees. Whereas SIV proteins had minor effects on the growth kinetics, MuV proteins substantially retarded the propagation of the respective viruses. Thus, the MV/MuV recombinants are not suitable as candidate vaccines. More generally, it may be concluded that for vaccination purposes it is not suitable to construct recombinants based on a paramyxovirus expressing additional components of another paramyxovirus. However, the construction of chimera in which components of one paramyxovirus are substituted by other paramyxovirus components [5] might be useful for particular purposes.
4. Discussion We have genetically engineered measles viruses for possible use as efficacious multivalent live-vaccine candidates. The results described here show that the non-segmented, negative-strand measles virus genome has the potential not only to stably express additional short or large heterologous transcripts of cytoplasmically expressed marker genes like CAT, GFP, and b-gal [4,17] or small virus-derived genes such as those encoding HBV c and s antigens [7], but also complete ORFs of viral genes exceeding 3 kb. Many viral systems have limitations in their ability to accept and stably maintain the expression of long transgenes. The successful rescue and subsequent spread of measles viruses expressing large transgenes over many rounds of replication suggest the utility of products as vaccine vectors, given the high efficacy and safety of attenuated MVs as vaccines. However, a limitation to this approach has been delineated here, specifically the interference of transgene from a closely related virus such as MuV, which was not observed in the case of unrelated viruses, specifically HBV as shown earlier [7] and SIV as shown here. Expression of the MuV virus surface glycoproteins in the presence of the normal complement of MV surface glycoproteins negatively affects the propagation kinetics of these particular rMVs, and results in a negative selection pressure. This can eventually lead to the interruption of such reading frames by spontaneous mutations, thus almost restoring the growth kinetics to that of standard MV, despite the maintenance of the large insert altered only by point mutations. Importantly, SIV protein expression was more stable, and affected the propagation of the cognate rMVs only slightly.
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Thus, the construction of rMVs carrying transgenes of other pathogens appears now promising. In particular, if macaques inoculated with the MV/SIV recombinants shown in this paper and with rMVs expressing nef and tat regulatory proteins (F. Tangy, Institut Pasteur, Paris, unpublished results) show strong immune responses and protection against lethal challenge with SIV, the construction and testing of analogous MV/HIV recombinants is warranted. Although no universal consensus exists on what components will be needed in a vaccine that is able to induce protective immunity against HIV-1 infection, such MV/HIV recombinants appear at least as appropriate as candidates using other vector systems. In fact, it might be necessary to utilize different vectors in successive boost vaccinations to obtain complete protection. The current view is that both the humoral and the cellular arms of the human immune system should be efficiently stimulated [4], which should be attainable using rMVs expressing a set of entire env, gag, pol, nef and tat proteins. The Edmonston B strain of measles is an approved, safe and efficacious vaccine that induces both long lasting, humoral and cellular immunity. The reason for this high efficacy and safety, even in comparison with the highly successful live polio vaccine is not understood so far, and might at least in part be due to the cell tropism, efficiently targeting antigen presenting cells such as macrophages and dendritic cells [2,18]. Thus, rMVs expressing antigens of very serious pathogens against which no vaccines exist so far, such as HIV, plasmodia eliciting Malaria and hepatitis C virus, might be particularly suitable to combat the corresponding diseases in developing countries. One negative aspect for the use of rMVs is the fact that in almost all individuals of the adult population there is pre-existing immunity against MV, due to either a history with acute measles or a measles vaccination in early childhood. However, the potential benefits of immunizing children early in life with a cocktail of rMVs expressing antigens of all major diseases rather than with a standard MV vaccine, as currently enforced world-wide by the WHO measles eradication campaign must not be ignored. In addition, it has been shown that school children, when reimmunized with MV, mount important recall immune responses [18], contrary to the lack of anti-MV immune induction exhibited by small infants containing a threshold level of maternal anti-MV antibodies [19]. The response of older children to repeat MV vaccinations in comparison to the lack of immunization response in small infants may be due to the immature immune system of the latter. Thus, it is conceivable that also in adults vaccination with rMV cocktails will induce at least partial immunity against the pathogens from which the transgenes inserted into the MV vector were derived. The
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particularly serious morbidity usually monitored in first infections by a particular pathogen might thereby be circumvented. Nevertheless, reasons for caution exist. First, measles induces strong immunosuppression, as does the attenuated standard MV vaccine albeit to a minor extent. The even more attenuated rMVs will probably also exhibit at least some of this property, which in the case of individuals having contracted HIV before could constitute a contraindication for the use of any live MV vaccine. Second, rMVs expressing envelope glycoproteins of other viruses might acquire a cell tropism differing from that of the standard live MV vaccines. This might be the case even if the foreign glycoprotein is not incorporated into MV particles, since MV spread within a host seems to occur primarily by cell/cell fusion [15,20,21]. Only experiments with primates will reveal whether these dangers are indeed serious in reality. In any event, rMVs could be constructed in which the fusion protein cannot be cleaved in the host, thus allowing in principle only one round of replication [22]. A drawback of such propagation-deficient rMVs would be the much larger doses required for each immunization, thus increasing the production cost and therefore the potential for the use of such vaccines in developing countries.
Acknowledgements Active collaboration with F. Tangy, Institut Pasteur, Paris, France, including exchange of results and materials is gratefully acknowledged. Plasmids bearing MuV and SIV genes were provided by Dr M.G. Cusi, Siena, Italy and by Dr R. Andino, San Francisco, CA, respectively. We thank Dr R. Glu¨ck of the Swiss Serum and Vaccine Institute (Berna) for his constant interest, discussions and support. This work was supported by grants from the Swiss National Science Foundation, 31.45900-95, the Commission for Technology and Innovation, 3786.1, and the NIH grant A146007-01.
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