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Production of Full-Length Human Monoclonal Antibodies using Transgenic Mice
CHAPTER TWENTY SIX
Production of Full-Length Human Monoclonal Antibodies using Transgenic Mice William D. Thomas Jr. MassBiologics of the University of Massachusetts Medical School, Boston, MA, USA
Chapter Contents 26.1 Introduction 317 26.2 Hybridoma Production 320 26.2.1 Mouse Immunization 320 26.2.2 Screening for Antibody Responses in Humanized Mice 321 26.2.3 Hybridoma Fusions 321 26.3 Hybridoma Screening and Characterization 323 26.3.1 Cell Culture and Screening 323 26.3.2 Virus Neutralization 324 26.3.3 Pseudovirus Neutralization 324 26.3.4 Epitope Mapping 325 26.3.5 Further Characterization 325 26.3.6 Hamster Post-Exposure Prophylaxis 326 26.4 Summary 326 References 327
26.1 INTRODUCTION Rabies is preventable when post-exposure prophylaxis (PEP) with vaccine and rabies immune globulin (RIG) is given in a timely fashion after exposure. However, there is still a significant amount of rabies mortality in endemic areas due to the unavailability and high cost of RIG. Neutralizing human monoclonal antibodies (HuMAbs) could be used for PEP affordably in these areas. The methods described here to isolate antibodies from transgenic mice with human antibody genes have identified a broadly neutralizing rabies HuMAb that is being tested clinically.1 Monoclonal antibodies (MAbs) intended for clinical use are ideally fully HuMAbs because of the advantages over murine, chimeric, or C. Rupprecht & T. Nagarajan (Eds): Current Laboratory Techniques in Rabies Diagnosis, Research and Prevention,Volume 2. Doi: http://dx.doi.org/10.1016/B978-0-12-801919-1.00026-9
© 2015 Elsevier Inc. All rights reserved.
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humanized antibodies. The HuMAbs are less likely to induce anti-drug antibodies (ADA) in the human host that could interfere with activity or half-life of the antibodies. The fully human antibodies should also be more effective in interactions with immune effector cells in treated individuals compared to murine antibodies.2 There are several technologies available to generate HuMAbs. A bestselling HuMAb therapeutic, adalimumab (Humira, AbbVie), used to treat rheumatoid arthritis and other autoimmune diseases,3 was isolated from human phage display libraries. Human immune globulin fragments are expressed on the surface of bacteriophages that carry the genes within the phage genome. Libraries containing large numbers of phage particles with different antibody specificities can be constructed. This is a very powerful technology because of the large diversity that can be created in phage libraries. Antibodies can be rapidly isolated by selecting phages that bind to an antigen of interest followed by sequencing of the antibody gene contained in the phage genome.4 Once isolated, a few rounds of antibody mutagenesis and panning can improve the affinity or activity of a given antibody.5 A comparable technology has been developed to create antibody libraries expressed on the surface of yeast cells.6 The yeast cells can be panned for binding antibodies, and their surfaces can be stained with antigens and sorted using a Fluorescence Activated Cell Sorter (FACS). The intensity of labeling determined by FACS analysis can be used to compare binding of antibodies early in the screening process.7 Construction steps required to produce phage libraries yield binding antibodies, but the process can produce molecules that are difficult to express or formulate in clinically relevant concentrations. Antibodies isolated from libraries need to be tested for “manufacturability” to confirm that they can be expressed, purified, and formulated in ways suitable for production.8 Human B cells can also be used to isolate HuMAbs. The HuMAbs can be obtained directly from human B cells by creating human hybridomas through Epstein–Barr Virus (EBV) immortalization.9 More recently, single-cell PCR for immune globulin genes from human B cells has also been used to isolate the HuMAbs. Single-cell PCR of sorted plasma cells obtained from immunized individuals has yielded MAbs to Tetanus and Diphtheria toxoids.10 Variable region genes are amplified by PCR and cloned into mammalian expression vectors with constant region genes to produce intact antibodies. One could also imagine obtaining useful HuMAbs from a convalescent individual following a similar strategy or
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by sorting memory B cells stained specifically with a relevant antigen.11 Antibody genes isolated directly from the cells of immune or convalescent individuals have been affinity maturated in humans and may represent an opportunity to isolate effective HuMAbs. Immunization of mice transgenic for human immune globulin genes followed by conventional murine hybridoma technology has also been used successfully to isolate HuMAbs.12 This chapter describes the methods used to isolate and characterize HuMAbs against rabies virus (RABV) from transgenic mice. A series of transgenes are required for a mouse to produce HuMAbs. First, these mice are defective in the production of murine antibodies. In the case of HuMAb mice (Bristol-Myers-Squib), this is accomplished with the introduction of deletions in the murine germ line sequences that prevent V-(D)-J gene rearrangements in the murine heavy and κ light chain genes. These mutations prevent the production of mouse heavy chains and κ light chains, but these mice still make murine λ light chains. This is not a significant issue because the human heavy chains rarely associate with murine λ light chains, and it is simple to confirm the presence of human κ light chains during screening of hybridomas by enzyme-linked immunosorbent assay (ELISA). Introduction of transgenes for the production of human heavy chains and human κ light chains was also done. A variety of human variable region transgenes have been introduced for human heavy chains and all the variable genes for human κ light chains. HuMAb mice are capable of germ line V-(D)-J rearrangements of human antibody genes, immune globulin class switching, and affinity maturation. The mice transgenes include the μ, γ1, and γ3 heavy chain constant regions and are capable of producing IgM, IgG1, and IgG3 isotype antibodies with κ light chains in their serum.13 Successful HuMAb selection strategies require strong supporting expertise in molecular immunology and molecular biology. The laboratory should have access to facilities for animal husbandry suitable for mice. Equipment for high-throughput ELISA analysis such as plate washers and readers facilitates the isolation process. Access to equipment for FACS analysis and affinity measurement, such as Surface Plasmon Resonance (SPR, Biacore, GE Healthcare) or Bio-Layer Interferometry (BLI, Octet, Pall) enables thorough characterization of HuMAbs. Expertise in modern molecular biology techniques and access to services such as DNA sequencing and gene synthesis is an advantage. Purification of recombinant proteins expressed in mammalian cells is important to efficient isolation of anti-viral MAbs. This is particularly true for RABV due to the
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inherent biosafety risks. Expression of RABV glycoprotein (G) in soluble forms enables screening of MAbs by ELISA and determination of affinity. Cell surface expression allows cell staining and pseudovirus production to investigate native confirmations of the G. Expression of variants of the G such as exotic RABV, escape mutants, and chimeric proteins can be used in epitope mapping and pseudovirus neutralization experiments.14 These experimental data will strengthen the arguments for the successful development of appropriately broadly neutralizing rabies HuMAbs.
26.2 HYBRIDOMA PRODUCTION 26.2.1 Mouse Immunization RABV-neutralizing epitopes are located in G, which is the major surface antigen, so whatever antigen/vaccine is used, it must include the G. The G should be prepared in such a way as to preserve the conformational epitopes that are likely to be the target of neutralizing antibodies. The soluble ectodomain (19–551 aa) of the Evelyn Rokitnicki Abelseth (ERA) G can be expressed in mammalian cells using commercially available vectors and purified to immunize mice.15 The soluble G can also be used as a coating antigen for screening of murine sera and hybridoma culture supernatants by ELISA. Commercial human rabies vaccines such as RabAvert (Novartis),16 Imovax (Sanofi),17 or Rabivax (Serum Institute of India)18 can be used for HuMAb mouse immunizations. Animals should be hyperimmunized over the course of several months to induce robust antibody responses before attempting to isolate hybridomas. If a purified ectodomain of G is used, each animal should be given doses in the range of 5–50 μg. Adjuvants such as aluminum hydroxide (Reheis) or RIBI (Sigma) should be mixed with soluble G. Doses should be prepared in a volume of up to 0.5 mL and administered via the intraperitoneal (IP) route. Booster doses should be given after the initial immunizations at intervals of ≥1 week. We have successfully used weekly doses, but a strategy with a longer duration between doses would also be effective. If commercially available inactivated vaccines are used, reconstitute the vaccine according to suppler instructions and administer a vaccine volume up to 0.5 mL by the IP route in mice. HuMAb mice were successfully immunized using a protocol that involved the use of 1/10 of a human dose of RabAvert™ (Novartis) or Imovax® (Aventis) rabies vaccines using complete Freund’s adjuvant in the first week, and RIBI
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adjuvant in subsequent weeks for a total of 6–8 weeks.15 Immune response in mice should be monitored using a serum ELISA to detect human antiRABV antibody response to immunization.
26.2.2 Screening for Antibody Responses in Humanized Mice A RABV G ELISA was used to initially screen murine sera and hybridoma cultures to determine whether they were producing anti-RABV G antibodies. Several sources of G can be used for the ELISA-based screening to demonstrate sero-conversion in immunized animals. The ELISA assay can be performed by coating plates with the same soluble G used to immunize the animals. Alternatively, concentrated stocks of inactivated whole virus can be coated on the plate. A commercially available RABV G ELISA (Platelia, BioRad) is also available to measure the antibody responses.19 These ELISA plates are developed using a polyclonal antihuman IgG-alkaline phosphatase conjugate. The soluble ectodomain of the RABV G can be coated onto a 96-well plate.15 To perform the ELISA, 96-well plates (Immulon, Costar) were coated overnight at 4°C with 1 μg/mL of the soluble ectodomain of the RABV G in phosphate buffered saline (PBS; pH 7.5). The coating solution was removed by washing with wash buffer (PBS; 0.5% Tween 80) and then blocked with blocking buffer (PBS; 1% BSA) at room temperature (RT) for one hour. Murine serum dilutions were prepared in blocking buffer, then added to the coated wells and incubated at RT for 1 hour. Serum samples were removed with wash buffer and the plate was developed using goat anti-human IgG-alkaline phosphatase conjugate (IgG-AP, Jackson ImmunoResearch). Finally, the plates were developed with p-nitrophenyl phosphate disodium salt at 1 mg/mL in 1 M diethanolamine for 20 minutes. The plates were read at 405 nm with an ELISA reader (Molecular Devices).
26.2.3 Hybridoma Fusions Mice with strong serum human IgG responses to the G were selected for cell fusion. Hybridomas were generated by fusion of mouse splenocytes and mouse myeloma cells followed by selection with hypoxanthineaminopterin-thymidine (HAT) medium. Mice were boosted intravenously through the tail vein with soluble antigen without adjuvant to induce B cell proliferation three days prior to isolation of splenocytes. If a particulate vaccine is the only immunogen available, mice can be boosted through the IP route. On the day of the fusion, animals were anesthetized with
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isoflurane inhalation or injected with ketamine/xylazine before euthanasia by cervical dislocation. The spleen was removed aseptically and splenocytes isolated by kneading the spleen between sterile forceps or sintered glass slides into Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin and streptomycin (pen-strep). Cells were harvested by centrifugation and the pellet re-suspended in ammonium chloride solution to lyse erythrocytes, and then diluted 1:4 with cold DMEM containing 1% penstrep. Splenocytes were harvested by centrifugation and washed twice with DMEM containing 1% pen-strep. Viable splenocytes were counted by the trypan blue dye exclusion principle. Depending on the animal and the size of the spleen, there should be 5–10 × 107 splenocytes available for fusion. The mouse myeloma fusion partner (P3X63Ag8.653) can be obtained from the American Type Culture Collection (ATCC) and maintained by standard cell culture techniques. Cells are cultured using RPMI (Fisher 10-040CM) supplemented with 1% pen-strep, L-glutamine, HEPES, pyruvate, 2-mercaptoethanol, and fetal bovine serum (FBS). Cells cultured in RPMI maintenance media at 37°C with 5% CO2 should divide every 24 hours. Myeloma cells should be split to a minimum density of 4 × 105 and grown to a maximum density of 2 × 106. Viability is important to successful hybridoma growth and should be >95% at the time of the fusion. Log-phase myeloma cells from four T-150 flasks (~2.0 × 108 total cells) are harvested by centrifugation and washed three times with DMEM containing 1% pen-strep and resuspended in 20 mL DMEM containing 1% pen-strep. Viable myeloma fusion partner cells are counted by the trypan blue dye exclusion principle. Cell fusion is accomplished by mixing splenocytes with myeloma cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG). For example, a fusion with the scale of twenty 96-well plates requires that 3 × 107 splenocytes are mixed with 1 × 107 myeloma cells and harvested by centrifugation. The cell pellet is gently re-suspended by flicking the tube several times and placing it in a water bath equilibrated to 37°C. Cells are fused using 1 mL of 50% PEG 1450 solution (Sigma, #7181) added dropwise over a minute. The fused cells are fragile so media additions should be done slowly with gentle mixing. After swirling, 4 mL of 37°C DMEM is gently added drop-wise over several minutes, then 40 mL of pre-warmed DMEM is added over several minutes at 37°C with gentle swirling. The fused cells are harvested by centrifugation with a slow spin using gentle acceleration to protect fragile hybrids. Aspirate the supernatant and gently re-suspend the pellet of fused cells in 30 mL of HAT selection medium. The HAT medium (Table 26.1) is based on DMEM supplemented with
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Table 26.1 HAT Selection Media Recipe for 500 mL Volume Component
Vendor, Catalog #
339.5 mL 5 mL 5 mL 5 mL 10 mL 10 mL 25 mL 0.5 mL 50 mL 50 mL
VWR, #45000-314 Fisher #MT-25-025-CI Fisher #MT-30-002-CI Sigma #O5003-1VL VWR #12001-698 Life Technologies #21060-017 Roche #11088947001 Life Technologies #21985-023 Life Technologies #21340-039 Lonza cat# 14-501 F
DMEM 100 × Non-Essential Amino Acids 100 × Penicillin-Streptomycin 100 × OPI supplement L-Glutamine 200 mM 50 × HAT BM Condimed H1 (20×) 1,000 × 2-Mercaptoethanol Medium NCTC-109 FBS – heat inactivated
non-essential amino acids, 1% pen-strep, L-glutamine, HAT supplement (Life Technologies, #21060-017), OPI supplement (Sigma, #O5003), BM condimed H1 (Roche, 11088947001), 2-mercaptoethanol, NCTC medium (Life Technologies, 21340-039) and FBS (Lonza #14-501F). Allow the cells to recover at 37°C for an hour in selection media followed by a further 10-fold dilution (1.3 × 105 cells/mL) gently in prewarmed HAT medium. This plating cell density and selection procedure will yield 50–90% growth, with most wells containing growth from a single hybridoma. Adjustment of the plating density may be required to optimize proportionate growth and clonality. Cell suspensions are plated in sterile 96-well flat bottom plates (Falcon, #35-3072) using wide bore pipette tips with 150 μL in each well. Plates are placed in a 37°C incubator with 5% CO2. Feed the fusion plates with 100 μL pre-warmed fresh HAT media after 7 days. Observe plates for contamination and clonal hybridoma growth, which should be visible within 7–10 days. On Day 14, the culture supernatant may be screened by a G ELISA.
26.3 HYBRIDOMA SCREENING AND CHARACTERIZATION 26.3.1 Cell Culture and Screening Hybridoma supernatants (100 μL) are screened for the reactivity of human MAb with RABV G as described for the serum ELISA above. Positive cultures are identified and transferred to fresh 96-well plates and fed with fresh HAT medium. A secondary screening of human MAbs by ELISA
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was done to confirm the results of primary screening and rule out the cross-reactivity with negative control antigens. The positive hybridomas are confirmed to have a human κ light chain by capture ELISA, which involves anti-human κ light chain (Southern Biotech) in the solid phase and developing the plate with goat anti-human IgG-AP conjugate (Jackson ImmunoResearch). The RNA from cultures that are positive for both the G ELISA and human κ light chain was extracted (RNAeasy 96 kit, Qiagen) and sent for cDNA sequencing of the human heavy chain variable region to identify sibling hybridomas (GeneWiz). The ELISAreactive hybridomas with unique variable heavy chain gene sequences are scaled up for further characterization by a virus neutralization test such as the Rapid Fluorescent Focus Inhibition Test (RFFIT), epitope mapping, and affinity determination. Hybridoma cultures typically express a range of antibody concentrations from 1–50 μg/mL. Cultures are expanded in HAT medium with gradually increasing volumes to produce mg quantities for characterization studies. The final expanded cultures (100–200 mL) are allowed to grow to low viability to maximize the expression of antibodies. After removal of cells by filtration, antibodies are purified from cell culture supernatants by protein A affinity chromatography for further characterization. Briefly, cells are removed from the culture supernatant by filtration and the culture supernatant applied to a protein A column (MabSelect, GE Healthcare) and eluted with 100 mM glycine buffer (pH 3). Purified antibodies are dialyzed against PBS and stored at 4°C.
26.3.2 Virus Neutralization Once hybridomas that have the specificity for RABV G as determined by the G ELISA have been isolated, the most critical test is to determine the ability to neutralize RABV. Positive hybridoma cultures are tested by the RFFIT against fixed RABV strains such as ERA or CVS11. The RFFIT is done by standard techniques (see Chapter 17). Purified antibodies can be titrated against the fixed RABV strains to rank the neutralization potency of the HuMAbs. Because broad neutralization is desired, additional RFFIT testing against a wider panel of RABV isolates should be done. The viruses included in the panel should be representative of the isolates from rabid animals in the specific geographic region intended for use of the antibodies.
26.3.3 Pseudovirus Neutralization Inhibition of G receptor binding can be accomplished by performing neutralization studies with RABV pseudovirus particles. Particles are produced by co-transfection of a replication-defective HIV backbone
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(Env-, Vpr-) carrying the firefly luciferase gene inserted into the nef gene, pNL4-3.Luc.R-E-, with RABV G encoding plasmids into 293T cells. Pseudoviral particles are harvested, concentrated, and frozen at −80°C. The luciferase counts per second (cps) of the pseudovirus preparations are determined by serial dilution of the virus followed by infection and detection using the Victor3 multilabel plate reader (Promega). For the neutralization assays, pseudoviruses are incubated with and without antibody for 1 hour at room temperature. The antibody/virus mix is then applied to HOS cells (ATCC# CRL-1543), in the presence of 2 μg/mL of polybrene and spinoculated for 2 hours at 800 g and 4°C, followed by incubation at 37°C/5% CO2. Luciferase activity was assayed 72 hours post-infection using the Bright-Glo reagent (Promega), according to the manufacturer’s protocol.15
26.3.4 Epitope Mapping Expression of fragments of the G ectodomain followed by ELISA or Western blotting can be used to map contiguous epitopes. The G fragments with affinity purification tags like His are expressed in Escherichia coli or human cells (293T) and purified by nickel affinity chromatography. These fragments are coated on plates for ELISA or run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for Western blot analysis. Conformational epitopes can also be mapped by isolation and sequencing of viral escape mutants. Escape mutants are selected by passaging RABV in the presence of a neutralizing HuMAb in permissive cells followed by fluorescent staining to detect virus replication. Sequencing the cDNA of the G of viruses capable of replication can identify changes in the amino acids of the epitope.15 Antibody epitopes can also be defined by competition with other antibodies in the panel. HuMAb competition for a common binding site can be measured by BLI analysis (Octet, Pall). The first antibody is bound to the Octet tip using protein A or anti-human IgG tips. Soluble G is bound to the first antibody, and then a second antibody is added. If the second antibody is incapable of binding, the two antibodies compete for the same epitope.10 This approach can be applied to a panel of antibodies to define epitopes and potentially identify antibodies that may work synergistically in a HuMAb mixture.
26.3.5 Further Characterization Mammalian cells transfected with the full-length G, including the transmembrane domain, express the G on the cell surface and can be used to
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measure binding to conformational epitopes that may not be present in soluble forms. The RABV-specific HuMAbs binding can be detected by the FACS analysis. Comparison of staining intensity of FACS-sorted cells can be used to rank the HuMAb binding. Furthermore, expression of full-length G from different RABV isolates can be screened for binding to cell surfaces to evaluate the potential for neutralizing a wide panel of RABV isolates. These data can be used to complement RFFIT and pseudovirus testing. Antibody affinity to the G can be determined by SPR, (Biacore, GE Healthcare) or BLI (Pall). For BLI, purified HuMAbs are bound to Octet tips and exposed to different concentrations of the ectodomain of soluble G. The G binding is measured at the different concentrations and the affinity calculated.10
26.3.6 Hamster Post-Exposure Prophylaxis Prevention of RABV infection should be confirmed in an in vivo model of PEP, such as has been described previously.20 Briefly, laboratory animals, such as Syrian hamsters, are infected with a RABV isolate in the right gastrocneimus muscle. Approximately 24 hours after inoculation, a HuMAb or HuMAb cocktail or commercial RIG is administered at the site of virus inoculation (i.e. right gastrocnemius) and 50 μL of commercial rabies vaccine is administered in the other (left) gastrocnemius muscle, distant to the site of inoculation, on Days 0, 3, 7, and 14 (and 28, if required). Animals are monitored for signs of rabies for up to 60 days. Effective HuMAbs should be equal to or more effective than RIG in protecting animals.15
26.4 SUMMARY Relevant HuMAbs can be isolated from transgenic mice by the methods described here. After developing robust responses in mice with G, hybridomas are produced by standard techniques, then screened by an ELISA and virus neutralization by RFFIT. The neutralizing HuMAbs are characterized to determine the breadth of neutralization, binding epitopes, affinity to G, and protection in animals to select the best antibodies for further development. The characterization of the antibody should demonstrate that geographically relevant RABV isolates are effectively neutralized by the HuMAb(s). This strategy was successfully used to identify and develop a HuMAb for human PEP that is in pivotal clinical trials in India to replace RIG (Clinical Trials Registry-India: CTRI/2012/05/002709).
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A HuMAb could also replace RIG in other parts of the world for rabies PEP. Where the diversity of endemic RABV presented a particular challenge, a mixture of several HuMAbs could be used to ensure that passive immune protection was sufficiently broad to protect PEP patients from the initiation of treatment until the development of a protective vaccine response.
REFERENCES 1. Gogtay N, Thatte U, Kshirsagar N, Leav B, Molrine D, Cheslock P, et al. Safety and pharmacokinetics of a human monoclonal antibody to rabies virus: a randomized, dose-escalation phase 1 study in adults. Vaccine 2012;30(50):7315–20. 2. Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332(6162):323–7. 3. Weinblatt ME, Keystone EC, Furst DE, Moreland LW, Weisman MH, Birbara CA, et al. Adalimumab, a fully human anti-tumor necrosis factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking concomitant methotrexate: the ARMADA trial. Arthritis Rheum 2003;48(1):35–45. 4. Lee CM, Iorno N, Sierro F, Christ D. Selection of human antibody fragments by phage display. Nat Protoc 2007;2(11):3001–8. 5. Holger T, Bernd V, Stefan D, Michael H, Thomas S. Affinity maturation by phage display. Methods Mol Biol 2009;525:309–22. 6. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD, et al. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 2006;1(2):755–68. 7. Achim D, Laura R, Stefan Z, Harald K. Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett 2014;588(2):278–87. 8. Xiaoyu Y, Wei X, Svetlana D, Sabrina B, Selina M, Valentyn A, et al. Developability studies before initiation of process development. MAbs 2013;5(5):787–94. 9. Schieffelin JS, Costin JM, Nicholson CO, Orgeron NM, Fontaine KA, Isern S, et al. Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient. Virology J 2010;7:28. 10. Sevigny LM, Booth BJ, Rowley KJ, Leav BA, Cheslock PS, Garrity KA, et al. Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication. Infect Immun 2013;81(11): 3992–4000. 11. Mouquet H, Klein F, Scheid JF, Warncke M, Pietzsch J, Oliveira TY, et al. Memory B cell antibodies to HIV-1 gp140 cloned from individuals infected with clade A and B viruses. PLoS One 2011;6(9):e24078. 12. Lonberg N. Fully human antibodies from transgenic mouse and phage display platforms. Curr Opin Immunol 2008;20(4):450–9. 13. Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, et al. Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994;368:856–9. 14. Wang Y, Rowley KJ, Booth BJ, Sloan SE, Ambrosino DM, Babcock GJ, et al. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates. Antiviral Res 2011;91(2):187–94. 15. Sloan SE, Hanlon C, Weldon W, Niezgoda M, Blanton J, Self J, et al. Identification and characterization of a human monoclonal antibody that potently neutralizes a broad panel of rabies virus isolates. Vaccine 2007;25(15):2800–10.
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16. RabAvert. Available from: [accessed 15.07.14]. 18. Rabivax. Available from:
Production of Full-Length Human Monoclonal Antibodies using Transgenic Mice William D. Thomas Jr. MassBiologics of the University of Massachusetts Medical School, Boston, MA, USA
Chapter Contents 26.1 Introduction 317 26.2 Hybridoma Production 320 26.2.1 Mouse Immunization 320 26.2.2 Screening for Antibody Responses in Humanized Mice 321 26.2.3 Hybridoma Fusions 321 26.3 Hybridoma Screening and Characterization 323 26.3.1 Cell Culture and Screening 323 26.3.2 Virus Neutralization 324 26.3.3 Pseudovirus Neutralization 324 26.3.4 Epitope Mapping 325 26.3.5 Further Characterization 325 26.3.6 Hamster Post-Exposure Prophylaxis 326 26.4 Summary 326 References 327
26.1 INTRODUCTION Rabies is preventable when post-exposure prophylaxis (PEP) with vaccine and rabies immune globulin (RIG) is given in a timely fashion after exposure. However, there is still a significant amount of rabies mortality in endemic areas due to the unavailability and high cost of RIG. Neutralizing human monoclonal antibodies (HuMAbs) could be used for PEP affordably in these areas. The methods described here to isolate antibodies from transgenic mice with human antibody genes have identified a broadly neutralizing rabies HuMAb that is being tested clinically.1 Monoclonal antibodies (MAbs) intended for clinical use are ideally fully HuMAbs because of the advantages over murine, chimeric, or C. Rupprecht & T. Nagarajan (Eds): Current Laboratory Techniques in Rabies Diagnosis, Research and Prevention,Volume 2. Doi: http://dx.doi.org/10.1016/B978-0-12-801919-1.00026-9
© 2015 Elsevier Inc. All rights reserved.
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humanized antibodies. The HuMAbs are less likely to induce anti-drug antibodies (ADA) in the human host that could interfere with activity or half-life of the antibodies. The fully human antibodies should also be more effective in interactions with immune effector cells in treated individuals compared to murine antibodies.2 There are several technologies available to generate HuMAbs. A bestselling HuMAb therapeutic, adalimumab (Humira, AbbVie), used to treat rheumatoid arthritis and other autoimmune diseases,3 was isolated from human phage display libraries. Human immune globulin fragments are expressed on the surface of bacteriophages that carry the genes within the phage genome. Libraries containing large numbers of phage particles with different antibody specificities can be constructed. This is a very powerful technology because of the large diversity that can be created in phage libraries. Antibodies can be rapidly isolated by selecting phages that bind to an antigen of interest followed by sequencing of the antibody gene contained in the phage genome.4 Once isolated, a few rounds of antibody mutagenesis and panning can improve the affinity or activity of a given antibody.5 A comparable technology has been developed to create antibody libraries expressed on the surface of yeast cells.6 The yeast cells can be panned for binding antibodies, and their surfaces can be stained with antigens and sorted using a Fluorescence Activated Cell Sorter (FACS). The intensity of labeling determined by FACS analysis can be used to compare binding of antibodies early in the screening process.7 Construction steps required to produce phage libraries yield binding antibodies, but the process can produce molecules that are difficult to express or formulate in clinically relevant concentrations. Antibodies isolated from libraries need to be tested for “manufacturability” to confirm that they can be expressed, purified, and formulated in ways suitable for production.8 Human B cells can also be used to isolate HuMAbs. The HuMAbs can be obtained directly from human B cells by creating human hybridomas through Epstein–Barr Virus (EBV) immortalization.9 More recently, single-cell PCR for immune globulin genes from human B cells has also been used to isolate the HuMAbs. Single-cell PCR of sorted plasma cells obtained from immunized individuals has yielded MAbs to Tetanus and Diphtheria toxoids.10 Variable region genes are amplified by PCR and cloned into mammalian expression vectors with constant region genes to produce intact antibodies. One could also imagine obtaining useful HuMAbs from a convalescent individual following a similar strategy or
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by sorting memory B cells stained specifically with a relevant antigen.11 Antibody genes isolated directly from the cells of immune or convalescent individuals have been affinity maturated in humans and may represent an opportunity to isolate effective HuMAbs. Immunization of mice transgenic for human immune globulin genes followed by conventional murine hybridoma technology has also been used successfully to isolate HuMAbs.12 This chapter describes the methods used to isolate and characterize HuMAbs against rabies virus (RABV) from transgenic mice. A series of transgenes are required for a mouse to produce HuMAbs. First, these mice are defective in the production of murine antibodies. In the case of HuMAb mice (Bristol-Myers-Squib), this is accomplished with the introduction of deletions in the murine germ line sequences that prevent V-(D)-J gene rearrangements in the murine heavy and κ light chain genes. These mutations prevent the production of mouse heavy chains and κ light chains, but these mice still make murine λ light chains. This is not a significant issue because the human heavy chains rarely associate with murine λ light chains, and it is simple to confirm the presence of human κ light chains during screening of hybridomas by enzyme-linked immunosorbent assay (ELISA). Introduction of transgenes for the production of human heavy chains and human κ light chains was also done. A variety of human variable region transgenes have been introduced for human heavy chains and all the variable genes for human κ light chains. HuMAb mice are capable of germ line V-(D)-J rearrangements of human antibody genes, immune globulin class switching, and affinity maturation. The mice transgenes include the μ, γ1, and γ3 heavy chain constant regions and are capable of producing IgM, IgG1, and IgG3 isotype antibodies with κ light chains in their serum.13 Successful HuMAb selection strategies require strong supporting expertise in molecular immunology and molecular biology. The laboratory should have access to facilities for animal husbandry suitable for mice. Equipment for high-throughput ELISA analysis such as plate washers and readers facilitates the isolation process. Access to equipment for FACS analysis and affinity measurement, such as Surface Plasmon Resonance (SPR, Biacore, GE Healthcare) or Bio-Layer Interferometry (BLI, Octet, Pall) enables thorough characterization of HuMAbs. Expertise in modern molecular biology techniques and access to services such as DNA sequencing and gene synthesis is an advantage. Purification of recombinant proteins expressed in mammalian cells is important to efficient isolation of anti-viral MAbs. This is particularly true for RABV due to the
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inherent biosafety risks. Expression of RABV glycoprotein (G) in soluble forms enables screening of MAbs by ELISA and determination of affinity. Cell surface expression allows cell staining and pseudovirus production to investigate native confirmations of the G. Expression of variants of the G such as exotic RABV, escape mutants, and chimeric proteins can be used in epitope mapping and pseudovirus neutralization experiments.14 These experimental data will strengthen the arguments for the successful development of appropriately broadly neutralizing rabies HuMAbs.
26.2 HYBRIDOMA PRODUCTION 26.2.1 Mouse Immunization RABV-neutralizing epitopes are located in G, which is the major surface antigen, so whatever antigen/vaccine is used, it must include the G. The G should be prepared in such a way as to preserve the conformational epitopes that are likely to be the target of neutralizing antibodies. The soluble ectodomain (19–551 aa) of the Evelyn Rokitnicki Abelseth (ERA) G can be expressed in mammalian cells using commercially available vectors and purified to immunize mice.15 The soluble G can also be used as a coating antigen for screening of murine sera and hybridoma culture supernatants by ELISA. Commercial human rabies vaccines such as RabAvert (Novartis),16 Imovax (Sanofi),17 or Rabivax (Serum Institute of India)18 can be used for HuMAb mouse immunizations. Animals should be hyperimmunized over the course of several months to induce robust antibody responses before attempting to isolate hybridomas. If a purified ectodomain of G is used, each animal should be given doses in the range of 5–50 μg. Adjuvants such as aluminum hydroxide (Reheis) or RIBI (Sigma) should be mixed with soluble G. Doses should be prepared in a volume of up to 0.5 mL and administered via the intraperitoneal (IP) route. Booster doses should be given after the initial immunizations at intervals of ≥1 week. We have successfully used weekly doses, but a strategy with a longer duration between doses would also be effective. If commercially available inactivated vaccines are used, reconstitute the vaccine according to suppler instructions and administer a vaccine volume up to 0.5 mL by the IP route in mice. HuMAb mice were successfully immunized using a protocol that involved the use of 1/10 of a human dose of RabAvert™ (Novartis) or Imovax® (Aventis) rabies vaccines using complete Freund’s adjuvant in the first week, and RIBI
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adjuvant in subsequent weeks for a total of 6–8 weeks.15 Immune response in mice should be monitored using a serum ELISA to detect human antiRABV antibody response to immunization.
26.2.2 Screening for Antibody Responses in Humanized Mice A RABV G ELISA was used to initially screen murine sera and hybridoma cultures to determine whether they were producing anti-RABV G antibodies. Several sources of G can be used for the ELISA-based screening to demonstrate sero-conversion in immunized animals. The ELISA assay can be performed by coating plates with the same soluble G used to immunize the animals. Alternatively, concentrated stocks of inactivated whole virus can be coated on the plate. A commercially available RABV G ELISA (Platelia, BioRad) is also available to measure the antibody responses.19 These ELISA plates are developed using a polyclonal antihuman IgG-alkaline phosphatase conjugate. The soluble ectodomain of the RABV G can be coated onto a 96-well plate.15 To perform the ELISA, 96-well plates (Immulon, Costar) were coated overnight at 4°C with 1 μg/mL of the soluble ectodomain of the RABV G in phosphate buffered saline (PBS; pH 7.5). The coating solution was removed by washing with wash buffer (PBS; 0.5% Tween 80) and then blocked with blocking buffer (PBS; 1% BSA) at room temperature (RT) for one hour. Murine serum dilutions were prepared in blocking buffer, then added to the coated wells and incubated at RT for 1 hour. Serum samples were removed with wash buffer and the plate was developed using goat anti-human IgG-alkaline phosphatase conjugate (IgG-AP, Jackson ImmunoResearch). Finally, the plates were developed with p-nitrophenyl phosphate disodium salt at 1 mg/mL in 1 M diethanolamine for 20 minutes. The plates were read at 405 nm with an ELISA reader (Molecular Devices).
26.2.3 Hybridoma Fusions Mice with strong serum human IgG responses to the G were selected for cell fusion. Hybridomas were generated by fusion of mouse splenocytes and mouse myeloma cells followed by selection with hypoxanthineaminopterin-thymidine (HAT) medium. Mice were boosted intravenously through the tail vein with soluble antigen without adjuvant to induce B cell proliferation three days prior to isolation of splenocytes. If a particulate vaccine is the only immunogen available, mice can be boosted through the IP route. On the day of the fusion, animals were anesthetized with
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isoflurane inhalation or injected with ketamine/xylazine before euthanasia by cervical dislocation. The spleen was removed aseptically and splenocytes isolated by kneading the spleen between sterile forceps or sintered glass slides into Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin and streptomycin (pen-strep). Cells were harvested by centrifugation and the pellet re-suspended in ammonium chloride solution to lyse erythrocytes, and then diluted 1:4 with cold DMEM containing 1% penstrep. Splenocytes were harvested by centrifugation and washed twice with DMEM containing 1% pen-strep. Viable splenocytes were counted by the trypan blue dye exclusion principle. Depending on the animal and the size of the spleen, there should be 5–10 × 107 splenocytes available for fusion. The mouse myeloma fusion partner (P3X63Ag8.653) can be obtained from the American Type Culture Collection (ATCC) and maintained by standard cell culture techniques. Cells are cultured using RPMI (Fisher 10-040CM) supplemented with 1% pen-strep, L-glutamine, HEPES, pyruvate, 2-mercaptoethanol, and fetal bovine serum (FBS). Cells cultured in RPMI maintenance media at 37°C with 5% CO2 should divide every 24 hours. Myeloma cells should be split to a minimum density of 4 × 105 and grown to a maximum density of 2 × 106. Viability is important to successful hybridoma growth and should be >95% at the time of the fusion. Log-phase myeloma cells from four T-150 flasks (~2.0 × 108 total cells) are harvested by centrifugation and washed three times with DMEM containing 1% pen-strep and resuspended in 20 mL DMEM containing 1% pen-strep. Viable myeloma fusion partner cells are counted by the trypan blue dye exclusion principle. Cell fusion is accomplished by mixing splenocytes with myeloma cells at a ratio of 3:1 in the presence of polyethylene glycol (PEG). For example, a fusion with the scale of twenty 96-well plates requires that 3 × 107 splenocytes are mixed with 1 × 107 myeloma cells and harvested by centrifugation. The cell pellet is gently re-suspended by flicking the tube several times and placing it in a water bath equilibrated to 37°C. Cells are fused using 1 mL of 50% PEG 1450 solution (Sigma, #7181) added dropwise over a minute. The fused cells are fragile so media additions should be done slowly with gentle mixing. After swirling, 4 mL of 37°C DMEM is gently added drop-wise over several minutes, then 40 mL of pre-warmed DMEM is added over several minutes at 37°C with gentle swirling. The fused cells are harvested by centrifugation with a slow spin using gentle acceleration to protect fragile hybrids. Aspirate the supernatant and gently re-suspend the pellet of fused cells in 30 mL of HAT selection medium. The HAT medium (Table 26.1) is based on DMEM supplemented with
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Table 26.1 HAT Selection Media Recipe for 500 mL Volume Component
Vendor, Catalog #
339.5 mL 5 mL 5 mL 5 mL 10 mL 10 mL 25 mL 0.5 mL 50 mL 50 mL
VWR, #45000-314 Fisher #MT-25-025-CI Fisher #MT-30-002-CI Sigma #O5003-1VL VWR #12001-698 Life Technologies #21060-017 Roche #11088947001 Life Technologies #21985-023 Life Technologies #21340-039 Lonza cat# 14-501 F
DMEM 100 × Non-Essential Amino Acids 100 × Penicillin-Streptomycin 100 × OPI supplement L-Glutamine 200 mM 50 × HAT BM Condimed H1 (20×) 1,000 × 2-Mercaptoethanol Medium NCTC-109 FBS – heat inactivated
non-essential amino acids, 1% pen-strep, L-glutamine, HAT supplement (Life Technologies, #21060-017), OPI supplement (Sigma, #O5003), BM condimed H1 (Roche, 11088947001), 2-mercaptoethanol, NCTC medium (Life Technologies, 21340-039) and FBS (Lonza #14-501F). Allow the cells to recover at 37°C for an hour in selection media followed by a further 10-fold dilution (1.3 × 105 cells/mL) gently in prewarmed HAT medium. This plating cell density and selection procedure will yield 50–90% growth, with most wells containing growth from a single hybridoma. Adjustment of the plating density may be required to optimize proportionate growth and clonality. Cell suspensions are plated in sterile 96-well flat bottom plates (Falcon, #35-3072) using wide bore pipette tips with 150 μL in each well. Plates are placed in a 37°C incubator with 5% CO2. Feed the fusion plates with 100 μL pre-warmed fresh HAT media after 7 days. Observe plates for contamination and clonal hybridoma growth, which should be visible within 7–10 days. On Day 14, the culture supernatant may be screened by a G ELISA.
26.3 HYBRIDOMA SCREENING AND CHARACTERIZATION 26.3.1 Cell Culture and Screening Hybridoma supernatants (100 μL) are screened for the reactivity of human MAb with RABV G as described for the serum ELISA above. Positive cultures are identified and transferred to fresh 96-well plates and fed with fresh HAT medium. A secondary screening of human MAbs by ELISA
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was done to confirm the results of primary screening and rule out the cross-reactivity with negative control antigens. The positive hybridomas are confirmed to have a human κ light chain by capture ELISA, which involves anti-human κ light chain (Southern Biotech) in the solid phase and developing the plate with goat anti-human IgG-AP conjugate (Jackson ImmunoResearch). The RNA from cultures that are positive for both the G ELISA and human κ light chain was extracted (RNAeasy 96 kit, Qiagen) and sent for cDNA sequencing of the human heavy chain variable region to identify sibling hybridomas (GeneWiz). The ELISAreactive hybridomas with unique variable heavy chain gene sequences are scaled up for further characterization by a virus neutralization test such as the Rapid Fluorescent Focus Inhibition Test (RFFIT), epitope mapping, and affinity determination. Hybridoma cultures typically express a range of antibody concentrations from 1–50 μg/mL. Cultures are expanded in HAT medium with gradually increasing volumes to produce mg quantities for characterization studies. The final expanded cultures (100–200 mL) are allowed to grow to low viability to maximize the expression of antibodies. After removal of cells by filtration, antibodies are purified from cell culture supernatants by protein A affinity chromatography for further characterization. Briefly, cells are removed from the culture supernatant by filtration and the culture supernatant applied to a protein A column (MabSelect, GE Healthcare) and eluted with 100 mM glycine buffer (pH 3). Purified antibodies are dialyzed against PBS and stored at 4°C.
26.3.2 Virus Neutralization Once hybridomas that have the specificity for RABV G as determined by the G ELISA have been isolated, the most critical test is to determine the ability to neutralize RABV. Positive hybridoma cultures are tested by the RFFIT against fixed RABV strains such as ERA or CVS11. The RFFIT is done by standard techniques (see Chapter 17). Purified antibodies can be titrated against the fixed RABV strains to rank the neutralization potency of the HuMAbs. Because broad neutralization is desired, additional RFFIT testing against a wider panel of RABV isolates should be done. The viruses included in the panel should be representative of the isolates from rabid animals in the specific geographic region intended for use of the antibodies.
26.3.3 Pseudovirus Neutralization Inhibition of G receptor binding can be accomplished by performing neutralization studies with RABV pseudovirus particles. Particles are produced by co-transfection of a replication-defective HIV backbone
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(Env-, Vpr-) carrying the firefly luciferase gene inserted into the nef gene, pNL4-3.Luc.R-E-, with RABV G encoding plasmids into 293T cells. Pseudoviral particles are harvested, concentrated, and frozen at −80°C. The luciferase counts per second (cps) of the pseudovirus preparations are determined by serial dilution of the virus followed by infection and detection using the Victor3 multilabel plate reader (Promega). For the neutralization assays, pseudoviruses are incubated with and without antibody for 1 hour at room temperature. The antibody/virus mix is then applied to HOS cells (ATCC# CRL-1543), in the presence of 2 μg/mL of polybrene and spinoculated for 2 hours at 800 g and 4°C, followed by incubation at 37°C/5% CO2. Luciferase activity was assayed 72 hours post-infection using the Bright-Glo reagent (Promega), according to the manufacturer’s protocol.15
26.3.4 Epitope Mapping Expression of fragments of the G ectodomain followed by ELISA or Western blotting can be used to map contiguous epitopes. The G fragments with affinity purification tags like His are expressed in Escherichia coli or human cells (293T) and purified by nickel affinity chromatography. These fragments are coated on plates for ELISA or run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for Western blot analysis. Conformational epitopes can also be mapped by isolation and sequencing of viral escape mutants. Escape mutants are selected by passaging RABV in the presence of a neutralizing HuMAb in permissive cells followed by fluorescent staining to detect virus replication. Sequencing the cDNA of the G of viruses capable of replication can identify changes in the amino acids of the epitope.15 Antibody epitopes can also be defined by competition with other antibodies in the panel. HuMAb competition for a common binding site can be measured by BLI analysis (Octet, Pall). The first antibody is bound to the Octet tip using protein A or anti-human IgG tips. Soluble G is bound to the first antibody, and then a second antibody is added. If the second antibody is incapable of binding, the two antibodies compete for the same epitope.10 This approach can be applied to a panel of antibodies to define epitopes and potentially identify antibodies that may work synergistically in a HuMAb mixture.
26.3.5 Further Characterization Mammalian cells transfected with the full-length G, including the transmembrane domain, express the G on the cell surface and can be used to
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measure binding to conformational epitopes that may not be present in soluble forms. The RABV-specific HuMAbs binding can be detected by the FACS analysis. Comparison of staining intensity of FACS-sorted cells can be used to rank the HuMAb binding. Furthermore, expression of full-length G from different RABV isolates can be screened for binding to cell surfaces to evaluate the potential for neutralizing a wide panel of RABV isolates. These data can be used to complement RFFIT and pseudovirus testing. Antibody affinity to the G can be determined by SPR, (Biacore, GE Healthcare) or BLI (Pall). For BLI, purified HuMAbs are bound to Octet tips and exposed to different concentrations of the ectodomain of soluble G. The G binding is measured at the different concentrations and the affinity calculated.10
26.3.6 Hamster Post-Exposure Prophylaxis Prevention of RABV infection should be confirmed in an in vivo model of PEP, such as has been described previously.20 Briefly, laboratory animals, such as Syrian hamsters, are infected with a RABV isolate in the right gastrocneimus muscle. Approximately 24 hours after inoculation, a HuMAb or HuMAb cocktail or commercial RIG is administered at the site of virus inoculation (i.e. right gastrocnemius) and 50 μL of commercial rabies vaccine is administered in the other (left) gastrocnemius muscle, distant to the site of inoculation, on Days 0, 3, 7, and 14 (and 28, if required). Animals are monitored for signs of rabies for up to 60 days. Effective HuMAbs should be equal to or more effective than RIG in protecting animals.15
26.4 SUMMARY Relevant HuMAbs can be isolated from transgenic mice by the methods described here. After developing robust responses in mice with G, hybridomas are produced by standard techniques, then screened by an ELISA and virus neutralization by RFFIT. The neutralizing HuMAbs are characterized to determine the breadth of neutralization, binding epitopes, affinity to G, and protection in animals to select the best antibodies for further development. The characterization of the antibody should demonstrate that geographically relevant RABV isolates are effectively neutralized by the HuMAb(s). This strategy was successfully used to identify and develop a HuMAb for human PEP that is in pivotal clinical trials in India to replace RIG (Clinical Trials Registry-India: CTRI/2012/05/002709).
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A HuMAb could also replace RIG in other parts of the world for rabies PEP. Where the diversity of endemic RABV presented a particular challenge, a mixture of several HuMAbs could be used to ensure that passive immune protection was sufficiently broad to protect PEP patients from the initiation of treatment until the development of a protective vaccine response.
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16. RabAvert. Available from: