key: cord-0864197-cmbg0ty8 authors: Mühlebach, Michael D.; Hutzler, Stefan title: Development of Recombinant Measles Virus-Based Vaccines date: 2016-11-26 journal: Recombinant Virus Vaccines DOI: 10.1007/978-1-4939-6869-5_9 sha: 3ff423438d2142229c5615b6ff80ed6d354fa81e doc_id: 864197 cord_uid: cmbg0ty8 This chapter describes the development of recombinant measles virus (MV)-based vaccines starting from plasmid DNA. Live-attenuated measles vaccines are very efficient and safe. Since the availability of a reverse genetic system to manipulate MV genomes and to generate respective recombinant viruses, a considerable number of recombinant viruses has been generated that present antigens of foreign pathogens during MV replication. Thereby, robust humoral and cellular immune responses can be induced, which have shown protective capacity in a substantial number of experiments. For this purpose, the foreign antigen-encoding genes are cloned into additional transcription units of plasmid based full-length MV vaccine strain genomes, which in turn are used to rescue recombinant MV by providing both full-length viral RNA genomes respective anti-genomes together with all protein components of the viral ribonucleoprotein complex after transient transfection of the so-called rescue cells. Infectious centers form among these transfected cells, which allow clonal isolation of single recombinant viruses that are subsequently amplified, characterized in vitro, and then evaluated for their immunogenicity in appropriate preclinical animal models. Vaccines are the most effective way to prevent infectious disease in terms of safety and cost-benefit ratio. However, at present, the development of vaccines to the point of licensing for human use takes decades and sometimes has proven hardly possible as exemplified by the HIV pandemic. To minimize the time for vaccine development and to be safe, it is necessary to develop strategies that allow for the immediate initiation of standardized vaccine development, leading to successful and safe candidate vaccines in a minimal amount of time. One strategy is to use well-known, already authorized vaccines with exceptional safety and efficacy records as a platform to present critical antigens of the pathogen which is the focus of vaccine development. Thereby, efficacious immune responses are triggered in immunized animals and patients not only against the vaccine vector, but also against the additionally present extra antigen. One of these potential vaccine platforms currently under development are recombinant vaccine strainderived measles viruses. Unmodified live-attenuated measles virus (MV) vaccine strains are efficient replicating vaccines. Besides revealing an excellent safety record, both humoral and cellular immune responses are elicited, which are responsible for long-lasting protection [1] [2] [3] . Therefore, the WHO targets eradication of measles by using these vaccines [4] . The vaccine's manufacturing process is extremely well established [5] and millions of doses can be generated quite easily and quickly. Generation of recombinant MV from DNA via reverse genetics became feasible [5] and allows for the robust expression of different antigens, as outlined below, during replication of the modified recombinant MV vaccine viruses. Thereby, generally robust immune responses against vector and foreign antigens are induced after vaccination of transgenic MV-susceptible IFNAR −/− -CD46Ge mice [6] , nonhuman primates, or human patients (see Table 1 ) indicating the high efficiency of the system. Interestingly, pre-vaccinated animals with protective immunity against measles were still amendable to vaccination with the recombinant MV, since significant immune responses against the foreign antigen(s) are still induced [7, 8] . Also in human probands, levels of antibodies against chikungunya virus (CHIKV) Env antigens did not display a negative correlation with preexisting antibody levels against the MV vector backbone [9] . A plethora of different antigens of various viruses and of one bacterium have been cloned into recombinant MV genomes, and the respective recombinant viruses have already been rescued. These projects are summarized in the following table (Table 1) . As depicted, mainly structural proteins, especially viral envelope glycoproteins have been expressed as antigens by the yet generated recombinant MV-derived vaccines, simply due to the expectation that especially humoral immunity against these antigens may result in neutralizing antibodies with protective capacity. However, the vector system proved also capable to induce robust cellular immune responses. Moreover, also secreted, cytoplasmic, or membrane bound markers, such as different luciferases, GFP, lacZ [13] , or cellular proteins such as carcinoembryonic antigen (CEA) [41] can be expressed by recombinant MV as demonstrated during its characterization and application as a potential oncolytic virus. Even membrane pore proteins such as the sodium iodide symporter (NIS) [42] and up to three different transgene cassettes with a size potentially exceeding 5 kb [13] have been successfully expressed by recombinant MV, demonstrating the general versatility of this vector system. The targeted viruses span a couple of genera and families. Also the developmental stage of the different vaccines is quite diverse, ranging from the demonstration of successful antigen expression by the recombinant MV, spanning the demonstration of humoral or cellular immunogenicity against the encoded antigen up to demonstration of the vaccine's protective efficacy in appropriate pre-clinical animal models. Of note, recombinant MV encoding the glycoprotein antigens of Chikungunya Virus, MV-CHIKV, has already been tested in a clinical phase I study in human volunteers [9] . After demonstrating efficacy in appropriate mouse and primate animal models [8] , this recombinant vaccine delivered proof of Triggered antigen-specific immune responses after immunization determined by measuring total antibodies (ELISA), neutralizing antibodies (nAbs), or reactive T cells determined by ELISpot or intracellular cytokine staining (ICS) c Protective capacity of vaccine-induced immune responses after challenge of the appropriate animal model determined by reduction of pathogen load or attenuation of etiopathology principle for safety and immunogenicity in human patients, irrespective of preformed anti-measles immunity [9] . Thus, the route for clinical development of such recombinant, MV-derived vaccines is open. On the one hand, these recombinant vaccines may be valuable to be used during primary measles vaccination to immunize children simultaneously against measles and a secondary pathogen of concern for the respective pediatric population without the need to vaccinate these children with two different vaccines. As an example, MV expressing hepatitis B virus small antigen (HBsAG) may be used in regions with high HBV prevalence to protect children early on from this potentially chronic infection ("buy one, get one free" strategy). On the other hand, recombinant MV is one of the potential vaccine platforms that can be used for the (fast track) development of vaccines against emerging pathogens, for which fast availability of a vaccine may be critical. In this respect, our MV-derived vaccine against the corona virus responsible for the Middle East Respiratory Syndrome (MERS-CoV) expressing the MERS-CoV glycoprotein S [38] has been among the first and most progressed vaccine candidates, which were evaluated by the Saudi Arabia Ministry of Health [43] and the WHO [44] . 1. Full-length MV genome plasmids such as pBR-MV vac2 -GFP(H) [10] or p(+)PolII-MV vac2 -ATU(P) [38] (see Note 1). 2. The MV genomes encoded on these plasmids contain the socalled additional transcription units (ATUs). To facilitate insertion of foreign ORFs, usually single-cutter restriction endonuclease recognition sites are placed between the start and stop signals of the ATU, in the above mentioned examples 5´ MluI and 3´ AatII (see Note 2). 3. T7-promoter driven expression plasmids for MV proteins being components of the viral RNP complex (i.e., nucleocapsid protein N, phosphoprotein P, viral RNA-dependent RNA-Polymerase L), such as pEMC-Na, pEMC-Pa, or pEMC-La [5, 45] , respectively. 5. cDNA of the foreign antigen to be expressed by the recombinant vaccine. The ORF has to be flanked 5´ by MluI and 3´ by AatII restriction sites (or alternative sites being part of the ATU) such that the genome length of the putative recombinant vaccines obeys to the "rule-of-six" [47] (see Note 3). 5. Cell are cultivated in DMEM supplemented with 10% fetal bovine serum and 2 mM L-Gln with additional 1.2 mg/mL of geneticin, when used for rescue as described in [5] . 6. All cells were cultured at 37 °C in a humidified atmosphere containing 6% CO 2 for no longer than 6 months after thawing of the original stock. 2. 30 G needles, one for each vaccine to be injected. 3. 2 mL syringes with fine scale, one for each vaccine to be injected. 1. Generation of gene segments encoding the desired foreign antigen of the target pathogen may be generated by gene synthesis. The (codon-optimized) ORF encompassing START and STOP codons is flanked by the respective restriction sites, which allow direct cloning of the gene segment into the ATU of a fulllength MV genome plasmid. It may be necessary to include a specific number of extra nucleotides between STOP codon and 3´restriction site to obey the rule-of-six (see Note 3). Alternatively, the desired ORF is amplified on the basis of (plasmid) cDNA or cDNA directly isolated (eventually after reverse transcription) from the target pathogens' genomes using (RT-)PCR. For this purpose, specific primers have to be designed as outlined above, depending on the exact structure of the ATU and the ORF to be amplified. 2. The resulting cDNA segments as well as the MV genome plasmids are then treated by the respective restriction endonucleases using the following standard reaction mix: 1-10 μg DNA. 5-10 U per restriction enzyme. 5 μl 10× NEB buffer (depending on the enzyme used). 5 μl 10× BSA (if required by the applied enzyme). Fill up to 50 μl with nuclease-free H 2 O. DNA digestions proceeds at the temperature required by the respective enzymes to allow thorough digestion of the DNA, optimally overnight. 3. The desired segments are purified after restriction digestion by standard agarose gel electrophoresis (see Note 4). The purified genome-containing plasmid backbone and the antigen-ORF containing insert are ligated using standard ligation conditions with at least tenfold molar excess of the insert: 50 ng vector DNA. 150 ng insert DNA. 2 μl 5× DNA dilution buffer. Fill up to 10 μl with nuclease-free water and mix. 10 μl 2× T4 DNA ligation buffer. 1. Seed 8 × 10 5 293T cells/well in 2 mL complete DMEM into six-well plates and incubate overnight (see Note 6). 2. The next day, cells should be approx. 80% confluent bevor starting transfection. 1. Seed 5 × 10 5 Vero cells/well in 2 mL complete DMEM into six-well plates and incubate overnight (see Note 6). 2. The next day, cells should be approx. 80% confluent bevor starting transfection. 3. Infection: Cells are infected with a T7-encoding vaccinia virus such as MVA-T7 at an MOI of 1-5 (see Note 10). 45 min after infection, the medium is replaced and cells are transfected. 4. Transfection: 1.5 μg of the respective MV genomic cDNA plasmid are mixed with 1.5 μg pEMC-Na, 1.5 μg pEMC-Pa, and 0.5 μg EMC.La (helper plasmids) and transfected using a commercially available lipofection method as described above in the protocol using the PolII-based rescue system. Transfected cells are cultured and examined for syncytia formation, daily (see Note 8). 1. If you find syncytia, pick them as outlined below (see Subheading 3.6, step 4). 2. If no syncytia are visible on day 4 post overlay, split the overlay culture by passaging the culture 1:4. For that purpose, seed 8 × 10 5 Vero cells/10 cm cell culture dish in 10 mL complete DMEM and incubate for 4 h. Aspirate the medium from the 10 cm cell culture dish of the overlay and wash once with 5 mL PBS. Detach the cells by incubating with 1.5 mL trypsin-EDTA for 5 min. After complete detaching (check by microscope) stop trypsin incubation by adding 2.5 mL complete DMEM and suspend the cells by pipetting up and down. Seed 25% of the cell suspension to the prepared 10 cm cell culture dish. The remaining 75% of the cell suspension are transferred into a 15 mL tube and snap-frozen in liquid nitrogen for 5 min. The frozen cell suspension is thawed at 37 °C in a water bath and the resulting cell debris is pelleted by centrifugation at 3000 × g for 5 min at 4 °C. Transfer the supernatant to the 10 cm cell culture dish dropwise and incubate overnight (see Note 11). If there is no syncytia formation wait for one additional day and if there is still no syncytia formation, repeat the rescue. 5. The cell suspension is transferred into precooled 1.5 mL reaction tubes and centrifuged at 17,000 × g for 15 min at 4 °C to remove the cell debris. 6. The protein containing supernatant is transferred into a fresh pre-cooled 1.5 mL reaction tube and stored at −80 °C. Frozen cell lysates are thawed on ice for further Western blot applications according to standard conditions using antibodies recognizing an MV protein, e.g., the nucleocapsid protein, to standardize for infection, and another antibody recognizing the foreign antigen to allow assessment of proper antigen expression by the recombinant vaccine. 4. Four to 7 days after booster immunization, splenozytes are harvested from immunized mice to assess abundance of antigen-or vector-specific T cells by ELISpot or intracellular cytokine staining after stimulation with respective antigens or peptides, either following standard protocols or using commercially available kits according to the manufacturer's instructions. 5. Before immunization ("pre-bleed"), directly before the booster vaccination ("post-prime") or 21 days after booster vaccination ("post-boost"), 200 μl blood are taken from each mouse, and sera are separated to assess abundance of vector-or target-specific antibodies by titrating neutralizing titers or total antibody titers by ELISA. 6. If the IFNAR −/− -CD46Ge mouse strain is susceptible to the pathogen to which the vaccine is directed against, vaccinated mice can be challenged subsequent to immunization. The challenge relies on an established infection protocol of (unvaccinated) animals, which results in symptomatic infections or even death, thereby allowing to test the protective capacity of the vaccine. Parameters such as course of disease or pathogen load in specific organs are assed (see Note 25). Both examples pBR-MV vac2 -GFP(H) and p(+)PolII-MV vac2 -ATU(P) contain, as all genome plasmids for the rescue of recombinant MV, a full-length MV vaccine strain genome. pBR-MV vac2 -GFP(H) is derived from the plasmid backbone pBR322 (low copy) and expresses the viral RNA antigenome under the control of a T7-promoter, whereas p(+)PolII-MV vac2 -ATU(P) has a pBluescript (high copy) backbone and is PolII-driven. Plasmids containing full-length MV genomes tend to be a bit delicate to handling procedures. Therefore, plasmids with a high-copy plasmid backbone should be amplified in E. coli at 30 °C, whereas low-copy plasmids can be amplified at 37 °C. In addition, it pays to directly pick clones or isolate plasmids from growing cultures instead of storing liquid bacteria cultures or colony plates with MV genomecontaining plasmids at 4 °C for more than few hours (≪ overnight!). As an alternative, bacteria pellets can be stored frozen at −20 °C before plasmid isolation. 2. Each MV gene cassette is flanked by conserved genetic elements representing start and stop signals for the viral polymerase complex. By duplicating these highly conserved intergenic sequences, a new transcription unit can be inserted in virtually any position of the virus genome, allowing insertion and expression of foreign genes such as antigen ORFs. The relative genomic position of the ATU allows regulation of the inserted gene's expression due to the transcriptional gradient found in Mononegavirales. The further upstream the ATU cassette is located, the higher the amount of mRNA being transcribed in infected cells and the higher protein expression. However, if the encoded gene product interferes with MV replication, too high expression levels of the encoded antigen may be detrimental. 3. The number of nucleotides in MV genomes can be exactly divided by 6, presumably due to one N protein binding to six nucleotides. Also recombinant genomes have to obey this rule; otherwise no recombinant virus can be rescued. Therefore, it has to be considered that the length of the inserted gene segment can be divided with the same rest by 6 as the length of the segment removed from the genome during cloning, if gene cassettes are inserted into genome plasmids. Thereby, one makes sure that the length of the resulting full-length genome is multiple of 6, again. 4. Due to the size of the genome-containing plasmid (approx. 20 kDa), voltage during agarose gel electrophoresis should be restricted to max. 70 V. 5. While T7-based rescue of MV, especially using the helper cell line 293-3-46, is the standard rescue system guaranteeing precise start and stop of the transcribed anti-genomic viral RNA due to the precise start of T7-driven transcription and stop due to specific termination signals in conjunction with the ribozyme flanking the viral genome sequences [5] , efficiency of the rescue is quite variable and depends considerably on the status of the rescue cells. Moreover, syncytia formation using 293-3-46 cells after overlay is not too efficient [5] , further limiting rescue efficiency especially of virus variants with limited fusion activity (unpublished own observation). Vaccinia Virus driven rescue allows usage of cell lines other than 293, which may be better suited for propagation of one specific MV variant, but depends on the quality of the used plasmids. PolII-driven virus rescue is very efficient and usually results in recombinant MV with precise genomes, but the lower precision of start and stop of Poll II transcription may allow completion of virus from genomes not being in line with the rule of six [46] . 6. Prepare one more six-well plate than required for the rescue of the different MV. This additional well is used as transfection control. 7. Seal six-well plate properly to avoid contamination of the cells. 8. If the recombinant MV will additionally express GFP or other fluorescent marker proteins, check for GFP-expression with the help of a fluorescence microscope to identify syncytia. If no easy marker protein for easy evaluation is encoded, check for syncytia formation via light microscope in phase contrast. 9. For transfection control, use 4 μg of for example EGFP expression plasmids such as pEGFP-N1 without any MV cDNA or helper-plasmids and transfect as described for MV cDNA. 10. If using another Vaccinia Virus than MVA for T7 Pol expression, one has to make sure that the rescued recombinant MV can be separated from (co-) replicating Vaccinia Virus, which may be a tedious process due to the laborious methods needed to physically separate the different virus populations. Step 2 seems to be slightly more efficient in our hands, but it includes one additional freeze-thaw cycle. 12. After quick-freezing in liquid nitrogen, the tube containing the virus suspension can be stored at −80 °C for several days. 13 . Check thawing of the virus regularly, the virus is heat sensitive and incubating the virus at 37 °C will lead to significantly reduced virus titers. 14. It is essential to slew the cell culture dish immediately as well as properly to avoid erratic infection of Vero cells, which will lead to lower virus titers. 15. The growth rate is depending on the virus strain and might be modulated by the additional genetic information. Therefore, check the virus growth regularly to develop a feeling for the growth rate. 16 . Scratch the cells to on edge of the cell culture dish and hold the plate inclined while aspirating the medium including all cell debris. 17. While MV vaccine strains authorized to be used as human vaccines are usually regarded as safe and accordingly being handled under BSL-1 conditions, recombinant viruses-even those bearing identical vaccine strain genomes-may be handled under BSL-1 or BSL-2 conditions depending on the locally responsible national or regional regulatory authorities. Under very rare circumstances, single recombinant MV may be placed into BSL-3, depending on the nature of the expressed foreign antigen. 18. Try to remove the medium completely while following Note 16. This will increase virus titers. 19. The number of aliquots can be modified, depending on freezerspace and experimental plans. 20. 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Cattaneo for introduction into the exciting field of recombinant MV and thank U. Schneider for providing the PolII Rescue System. The project was supported by Federal ministry for Education and Research of Germany (BMBF) grant 031A010B (M.D. Mühlebach).