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• W2743687550 abstract "Antibodies are the principal immune effectors that mediate protection against reinfection following viral infection or vaccination. Robust techniques for human mAb isolation have been developed in the last decade. The study of human mAbs isolated from subjects with prior immunity has become a mainstay for rational structure-based, next-generation vaccine development. The plethora of detailed molecular and genetic studies coupling the structure of antigen-antibody complexes with their antiviral function has begun to reveal common principles of critical interactions on which we can build better vaccines and therapeutic antibodies. This review outlines the approaches to isolating and studying human antiviral mAbs and discusses the common principles underlying the basis for their activity. This review also examines progress toward the goal of achieving a comprehensive understanding of the chemical and physical basis for molecular recognition of viral surface proteins in order to build predictive molecular models that can be used for vaccine design. Antibodies are the principal immune effectors that mediate protection against reinfection following viral infection or vaccination. Robust techniques for human mAb isolation have been developed in the last decade. The study of human mAbs isolated from subjects with prior immunity has become a mainstay for rational structure-based, next-generation vaccine development. The plethora of detailed molecular and genetic studies coupling the structure of antigen-antibody complexes with their antiviral function has begun to reveal common principles of critical interactions on which we can build better vaccines and therapeutic antibodies. This review outlines the approaches to isolating and studying human antiviral mAbs and discusses the common principles underlying the basis for their activity. This review also examines progress toward the goal of achieving a comprehensive understanding of the chemical and physical basis for molecular recognition of viral surface proteins in order to build predictive molecular models that can be used for vaccine design. A method for generating large amounts of murine mAbs of a predefined specificity was first published in 1975 by César Milstein and Georges J.F. Köhler (Köhler and Milstein, 1975Köhler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.Nature. 1975; 256: 495-497Crossref PubMed Google Scholar), who won the 1984 Nobel Prize in Physiology or Medicine for this advance. This laboratory method, creation of hybridoma cell lines, has revolutionized many fields of biomedical research including diagnostics and the generation of therapeutic antibodies for cancer, inflammation, immunotherapy, and infectious diseases. Increasingly, however, it has become apparent that murine antibodies are limited in their relevance for the very detailed types of structure/function studies that are needed for the design of next-generation human vaccines. Deep-sequencing studies are revealing that murine antibody variable gene repertoires are less complex and less diverse than those of humans. The average length of the most variable loop of the antigen-combining surface (the heavy-chain complementarity-determining region 3, or HCDR3) is shorter in mice than in humans (Shi et al., 2014Shi B. Ma L. He X. Wang X. Wang P. Zhou L. Yao X. Comparative analysis of human and mouse immunoglobulin variable heavy regions from IMGT/LIGM-DB with IMGT/HighV-QUEST.Theor. Biol. Med. Model. 2014; 11: 30Crossref PubMed Scopus (10) Google Scholar). Long HCDR3 regions are especially important in many antiviral antibody responses. The genetics of the Ig locus of experimental animals has homology with that of humans in many cases, but the actual genomic sequences differ significantly. In some cases, animal germline gene segments do not encode the specific sequences needed to bind to certain antigens. For example, the VRC01 class of HIV CD4-binding-site-specific neutralizing antibodies is not made in mouse or even monkeys, because specific sequences of the human VH1-02 antibody variable gene segment are needed for binding that do not occur in these animals (Navis et al., 2014Navis M. Tran K. Bale S. Phad G.E. Guenaga J. Wilson R. Soldemo M. McKee K. Sundling C. Mascola J. et al.HIV-1 receptor binding site-directed antibodies using a VH1-2 gene segment orthologue are activated by Env trimer immunization.PLoS Pathog. 2014; 10: e1004337Crossref PubMed Scopus (0) Google Scholar). The rhesus macaque VH1.23 gene segment is the closest primate ortholog to the human VH1-02 gene segment (exhibiting 92% homology to the VH1-02∗02 gene). Yet even the macaque VH1.23 alleles encode only two of the three amino acids in the human gene segment that define the critical motif for VRC01-like antibodies (W50, N58 flanking the HCDR2 region, and R71). In this review, first I will discuss the diverse laboratory techniques for isolation of human mAbs, the assays for measuring antiviral function of antibodies, and the relationship of neutralization activity to protection in vivo. Then, I present a review of the fundamental principles underlying the structural and genetic basis for development of antibodies that possess the desirable features of breadth and potency. Finally, I discuss the potential impact of our growing understanding of canonical features of protective human mAbs on the rational design of next-generation vaccines. The first several decades of hybridoma work focused on the isolation of murine mAbs for several reasons. First, mice can be repetitively infected or hyper-immunized with foreign antigens, to increase the frequency of antigen-specific B cells. Second, animals can be brought to necropsy to collect the spleen, which is a B cell-rich tissue that typically contains about 108 cells. In contrast, a typical 50-mL human peripheral blood sample for research contains only about 50 million mononuclear cells (of which only about 5%, or 2–3 million, are B cells). Third, the non-secreting myeloma fusion partners used in fusion with B cells worked well for murine cells, but human fusion partners were lacking. Eventually, investigators took on the task of developing techniques for efficient isolation of mAbs from human cells. First, investigators developed methods for cloning antibody variable genes by isolation of RNA followed by RT-PCR (Larrick et al., 1989Larrick J.W. Danielsson L. Brenner C.A. Abrahamson M. Fry K.E. Borrebaeck C.A. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction.Biochem. Biophys. Res. Commun. 1989; 160: 1250-1256Crossref PubMed Google Scholar). Once it was possible to obtain diverse amplicon libraries of variable genes, the logical next step was to clone and express the antibodies in phage display libraries that express the antibodies on the surface of phage particles and also package the variable gene cDNAs in phagemids inside the particles (Kang et al., 1991Kang A.S. Barbas C.F. Janda K.D. Benkovic S.J. Lerner R.A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces.Proc. Natl. Acad. Sci. USA. 1991; 88: 4363-4366Crossref PubMed Google Scholar, McCafferty et al., 1990McCafferty J. Griffiths A.D. Winter G. Chiswell D.J. Phage antibodies: filamentous phage displaying antibody variable domains.Nature. 1990; 348: 552-554Crossref PubMed Scopus (0) Google Scholar, Szardenings and Collins, 1990Szardenings M. Collins J. A phasmid optimised for protein design projects: pMAMPF.Gene. 1990; 94: 1-7Crossref PubMed Scopus (8) Google Scholar). We learned many important principles from studies in the early 1990s with human mAbs isolated from such libraries, including the surprising finding that Fabs alone could potently inhibit a virus replication program (Barbas et al., 1992Barbas 3rd, C.F. Crowe Jr., J.E. Cababa D. Jones T.M. Zebedee S.L. Murphy B.R. Chanock R.M. Burton D.R. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity.Proc. Natl. Acad. Sci. USA. 1992; 89: 10164-10168Crossref PubMed Google Scholar, Crowe et al., 1994Crowe Jr., J.E. Murphy B.R. Chanock R.M. Williamson R.A. Barbas 3rd, C.F. Burton D.R. Recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice.Proc. Natl. Acad. Sci. USA. 1994; 91: 1386-1390Crossref PubMed Google Scholar). Over time, however, it became apparent that the heavy- and light-chain pairing in such libraries is essentially random, and investigators sought new methods that could isolate antibodies from single cells with the natural pairing of heavy and light chains. Techniques for physical selection of single antigen-specific B cells were developed, but initially these methods were very inefficient. Antigen labeling of B cells by binding of purified or recombinant antigens to the B cell receptor (membrane-bound surface immunoglobulin) enabled single-cell flow cytometric sorting as the sorting instruments became more precise. Sorted single cells were used directly to obtain antibody genes (Babcook et al., 1996Babcook J.S. Leslie K.B. Olsen O.A. Salmon R.A. Schrader J.W. A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.Proc. Natl. Acad. Sci. USA. 1996; 93: 7843-7848Crossref PubMed Scopus (85) Google Scholar), or sorted single cells were expanded on feeder cell layers followed by RNA extraction and RT-PCR (Weitkamp et al., 2003Weitkamp J.H. Kallewaard N. Kusuhara K. Feigelstock D. Feng N. Greenberg H.B. Crowe Jr., J.E. Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.J. Immunol. Methods. 2003; 275: 223-237Crossref PubMed Scopus (44) Google Scholar). The emergence of this technique was exciting, but it was difficult to label and identify antigen-specific cells when the nonspecific binding of the viral reagents to all B cells was high in the setting of low frequency of B cell precursors in peripheral blood samples of many donors. Also, in order to identify antibodies that bind to the virion particles, the antigen used to sort cells must be in the correct conformation, with a high level of structural fidelity to the form of the protein on the virus. This feature is usually not the case with soluble forms of integral membrane proteins. For instance, hundreds of human mAbs were identified using the soluble form of the dengue envelope protein as a screening target, but very few of them demonstrated neutralizing activity (Smith et al., 2012Smith S.A. Zhou Y. Olivarez N.P. Broadwater A.H. de Silva A.M. Crowe Jr., J.E. Persistence of circulating memory B cell clones with potential for dengue virus disease enhancement for decades following infection.J. Virol. 2012; 86: 2665-2675Crossref PubMed Scopus (79) Google Scholar, Smith et al., 2013Smith S.A. de Alwis R. Kose N. Durbin A.P. Whitehead S.S. de Silva A.M. Crowe Jr., J.E. Human monoclonal antibodies derived from memory B cells following live attenuated dengue virus vaccination or natural infection exhibit similar characteristics.J. Infect. Dis. 2013; 207: 1898-1908Crossref PubMed Scopus (0) Google Scholar). However, a few of the antibodies that bound to soluble envelope proteins also could bind to live virus particles on immunoassay plates, suggesting that the recombinant protein was altered in some way compared to the virion protein. Subsequently, live virus particles that had been purified on density gradients (and in some cases additionally immunoaffinity purified by capture with antibodies) on ELISA plates served as a much more effective screening antigen to identify neutralizing mAbs (Smith et al., 2014Smith S.A. de Alwis A.R. Kose N. Jadi R.S. de Silva A.M. Crowe Jr., J.E. Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones.J. Virol. 2014; 88: 12233-12241Crossref PubMed Scopus (0) Google Scholar). Another method for isolating antigen-specific human B cells was Epstein-Barr virus (EBV)-mediated B cell transformation, which was used to activate and transform primary B cells in culture via the EBV receptor CD21 on some B cells. Initially, however, it was found that this technique generated mostly polyreactive antibodies of the IgM isotype. Subsequently, the addition of the TLR9 agonist CPG with EBV was shown to enhance transformation of a broader array of B cells (Bernasconi et al., 2002Bernasconi N.L. Traggiai E. Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells.Science. 2002; 298: 2199-2202Crossref PubMed Scopus (0) Google Scholar). Addition of cyclosporine to inhibit rapidly dividing EBV-specific CD8+ cytolytic cells in the PBMC sample (that otherwise lyse EBV peptide-presenting B cells) preserves the B cell cultures longer. A number of important human mAbs to SARS (Traggiai et al., 2004Traggiai E. Becker S. Subbarao K. Kolesnikova L. Uematsu Y. Gismondo M.R. Murphy B.R. Rappuoli R. Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus.Nat. Med. 2004; 10: 871-875Crossref PubMed Scopus (0) Google Scholar), CMV (Macagno et al., 2010Macagno A. Bernasconi N.L. Vanzetta F. Dander E. Sarasini A. Revello M.G. Gerna G. Sallusto F. Lanzavecchia A. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex.J. Virol. 2010; 84: 1005-1013Crossref PubMed Scopus (188) Google Scholar), paramyxoviruses (Corti et al., 2013Corti D. Bianchi S. Vanzetta F. Minola A. Perez L. Agatic G. Guarino B. Silacci C. Marcandalli J. Marsland B.J. et al.Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.Nature. 2013; 501: 439-443Crossref PubMed Scopus (103) Google Scholar, Wen et al., 2017Wen X. Mousa J.J. Bates J.T. Lamb R.A. Crowe Jr., J.E. Jardetzky T.S. Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus.Nat. Microbiol. 2017; 2: 16272Crossref PubMed Scopus (8) Google Scholar), and others have been derived with this technique. The transformation protocol was modified further to include an apoptosis inhibitor, specifically a small-molecule Chk2 inhibitor (Smith and Crowe, 2015Smith S.A. Crowe Jr., J.E. Use of human hybridoma technology to isolate human monoclonal antibodies.Microbiol. Spectr. 2015; 3: AID-0027-AID-2014Crossref Scopus (12) Google Scholar). Using this multipronged approach, a high efficiency of EBV transformation can be achieved. During this procedure, primary B cells proliferate and differentiate, secreting antibodies into the supernatants of the cell culture. EBV-transformed lymphoblastoid cell lines (LCLs) are difficult to clone biologically by passaging at the single-cell level, however. A combined transformation and hybridoma approach enables the rescue of unstable LCLs using electrofusion with a myeloma partner (Yu et al., 2008aYu X. McGraw P.A. House F.S. Crowe Jr., J.E. An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies.J. Immunol. Methods. 2008; 336: 142-151Crossref PubMed Scopus (75) Google Scholar). In this technique, the surface of LCLs and myeloma cells in suspension can be physically apposed using dielectrophoresis by delivering a low alternating current (Figure 1A), followed by direct current pulses that cause cell fusion. The human hybridoma workflow is summarized in Figure 1B. The resulting human hybridoma cells secreting mAbs can be cloned biologically more easily than LCLs (which often stop secreting the antibody of interest in low-cell-density culture) by limiting dilution or flow cytometric sorting. This approach has proven to be exceedingly robust, allowing isolation of very rare antigen-specific B cells from peripheral blood samples of humans infected with particular agents, decades after exposure by natural infection or immunization. For example, human mAbs that neutralized 1918 influenza A H1N1 virus were isolated from subjects who were ∼100 years of age in 2007, at a time when 1918-like viruses had not circulated for about half a century (Yu et al., 2008bYu X. Tsibane T. McGraw P.A. House F.S. Keefer C.J. Hicar M.D. Tumpey T.M. Pappas C. Perrone L.A. Martinez O. et al.Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors.Nature. 2008; 455: 532-536Crossref PubMed Scopus (244) Google Scholar). The same approach has been used for isolating human mAbs to dengue virus, using the blood of U.S.-dwelling subjects who had previous dengue virus infection acquired during foreign travel decades before (Smith et al., 2012Smith S.A. Zhou Y. Olivarez N.P. Broadwater A.H. de Silva A.M. Crowe Jr., J.E. Persistence of circulating memory B cell clones with potential for dengue virus disease enhancement for decades following infection.J. Virol. 2012; 86: 2665-2675Crossref PubMed Scopus (79) Google Scholar). Additional methods for human B cell isolation and mAb generation have continued to emerge. Different methods for activation and transformation of human B cells have been reported. For example, lentivirus-mediated transduction of memory B cells with two anti-apoptotic cellular factors, BCL6 and Bcl-xL, has been used to immortalize cells for mAb discovery (Kwakkenbos et al., 2010Kwakkenbos M.J. Diehl S.A. Yasuda E. Bakker A.Q. van Geelen C.M. Lukens M.V. van Bleek G.M. Widjojoatmodjo M.N. Bogers W.M. Mei H. et al.Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.Nat. Med. 2010; 16: 123-128Crossref PubMed Scopus (127) Google Scholar). The TLR7/8 agonist R848 (resiquimod, an imidazoquinoline compound) combined with IL-2 activates memory B cells (Pinna et al., 2009Pinna D. Corti D. Jarrossay D. Sallusto F. Lanzavecchia A. Clonal dissection of the human memory B-cell repertoire following infection and vaccination.Eur. J. Immunol. 2009; 39: 1260-1270Crossref PubMed Scopus (94) Google Scholar). IL-21 is a potent stimulator of CD40 ligand (CD154)-activated B cells, and B cell-activating factor (BAFF) enhances the stability of antibody-secreting cells. Combinations of these factors can be used to activate and maintain B cell lines. Another exciting development in the field was the discovery that antigen-specific B cells circulate as plasmablasts in the peripheral blood during a very circumscribed period following vaccination (about days 5–7) (Wrammert et al., 2008Wrammert J. Smith K. Miller J. Langley W.A. Kokko K. Larsen C. Zheng N.Y. Mays I. Garman L. Helms C. et al.Rapid cloning of high-affinity human monoclonal antibodies against influenza virus.Nature. 2008; 453: 667-671Crossref PubMed Scopus (544) Google Scholar). Plasmablasts are not common in the circulation of healthy subjects, but their frequency can exceed 1% of circulating B cells a week after vaccination. This finding was useful because in this setting antigen labeling of B cells (with its attendant problems of nonspecific binding) is not required, but instead one can sort all of the circulating plasmablasts and infer that they are specific for antigens from the recent immunization or infection. This method routinely yields interesting human mAbs. However, it is unclear at this time whether the dominant antibody clones in the plasmablast population persist into the B cell memory population in humans. Finally, investigators have raised the concern that studying only the human B cells in circulation may be limiting, since the secreted antibody proteins in circulation that mediate protection derive from long-lived plasma cells in the bone marrow. Some studies suggest the repertoire of the bone marrow plasma cells and circulating memory B cells differs (Purtha et al., 2011Purtha W.E. Tedder T.F. Johnson S. Bhattacharya D. Diamond M.S. Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants.J. Exp. Med. 2011; 208: 2599-2606Crossref PubMed Scopus (72) Google Scholar). Various approaches are being used to address this concern, including mass spectrometry-based sequencing of antigen-specific antibody proteins captured from serum or plasma (Boutz et al., 2014Boutz D.R. Horton A.P. Wine Y. Lavinder J.J. Georgiou G. Marcotte E.M. Proteomic identification of monoclonal antibodies from serum.Anal. Chem. 2014; 86: 4758-4766Crossref PubMed Google Scholar) and direct study of human bone marrow specimens. Antibody-mediated inhibition of viruses to define neutralization activity typically is measured by specific in vitro laboratory assays (although in vivo challenge studies also are used in some cases). Usually, live virus suspensions of known infectious particle count are mixed with varying concentrations of purified antibodies and incubated for a short period of time, then the mixtures are inoculated onto cell cultures to detect if any residual live virus exists. The readout is the percent reduction of infectious virus particles present in the initial inoculum. Many viruses can be detected using a plaque assay in a cell culture monolayer, such as poxviruses or paramyxoviruses like respiratory syncytial virus (RSV) (Figure 1C). This assay format is used to increase throughput and simplicity. In this approach, residual virus following incubation with antibodies is detected in cells that are inoculated in liquid medium, allowing virus to spread in culture, instead of the use of a semisolid or agar overlay that limits local spread and causes plaque formation. In the liquid overlay assay, detection of residual replication-competent virus is performed with detection of a viral antigen and chemical substrate using a colorimetric optical density in the well, which correlates with the amount of viral antigen in the cell monolayer. Various types of microneutralization assays can be compared for benchmarking and standardization, e.g., influenza assays being tested by the international partnership CONSISE (the Consortium for the Standardization of Influenza Seroepidemiology) (Laurie et al., 2015Laurie K.L. Engelhardt O.G. Wood J. Heath A. Katz J.M. Peiris M. Hoschler K. Hungnes O. Zhang W. Van Kerkhove M.D. CONSISE Laboratory Working Group participantsInternational laboratory comparison of influenza microneutralization assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) influenza viruses by CONSISE.Clin. Vaccine Immunol. 2015; 22: 957-964Crossref PubMed Scopus (3) Google Scholar). Specific mechanistic assays have been developed to study the particular step in the virus life cycle at which the antibodies are acting. For instance, some of the most potent virus-inhibiting antibodies prevent attachment to cell surface receptors. Blocking attachment to a recombinant or purified form of cell receptor can be used in a multi-well plate assay format even when the virus cannot be propagated, such as for noroviruses that attach to histoblood group antigens (HBGAs). In this system, virus-like particles (VLPs) are used to attach to purified HBGAs, and antibodies that block binding of VLPs to HBGA are considered effectively neutralizing. Norovirus does not replicate in conventional cultured cells, although a recent report describes replication of some human noroviruses in stem cell-derived human enteroids (Ettayebi et al., 2016Ettayebi K. Crawford S.E. Murakami K. Broughman J.R. Karandikar U. Tenge V.R. Neill F.H. Blutt S.E. Zeng X.L. Qu L. et al.Replication of human noroviruses in stem cell-derived human enteroids.Science. 2016; 353: 1387-1393Crossref PubMed Scopus (232) Google Scholar). The same principle of receptor blocking assay as a surrogate for neutralization also is used for hemagglutination inhibition for viruses that bind to sialic acid, such as influenza viruses. Sialic acid molecules are common on airway epithelial cells and also on the surface of red blood cells (RBCs). Therefore, virus agglutination of RBCs can be used as a surrogate of virus attachment to respiratory epithelial cells. Antibodies that block virus-mediated agglutination of RBCs in vitro are termed hemagglutination-inhibiting (HAI) antibodies. HAI antibody titers have been validated as a correlate of protection by regulatory agencies for release of influenza vaccines (U.S.F.D.A., 2007U.S.F.D.A. (2007). Guidance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines, F.D.A. U.S. Department of Health and Human Services, Center for Biologics Evaluation and Research. https://www.fda.gov/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/vaccines/ucm074794.htm.Google Scholar). Variations of this technique include systems using green fluorescent protein, luciferase, or other reporter genes. These approaches are particularly helpful for standardization using validated assays and standardized reference strains in a GCLP-compliant environment, such as has been done in the HIV field with the TZM-bl assay or A3R5 assay systems (Binley et al., 2004Binley J.M. Wrin T. Korber B. Zwick M.B. Wang M. Chappey C. Stiegler G. Kunert R. Zolla-Pazner S. Katinger H. et al.Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies.J. Virol. 2004; 78: 13232-13252Crossref PubMed Scopus (600) Google Scholar, Sarzotti-Kelsoe et al., 2014Sarzotti-Kelsoe M. Bailer R.T. Turk E. Lin C.L. Bilska M. Greene K.M. Gao H. Todd C.A. Ozaki D.A. Seaman M.S. et al.Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.J. Immunol. Methods. 2014; 409: 131-146Crossref PubMed Scopus (93) Google Scholar). In the TZM-bl assay, engineered pseudovirions displaying an envelope (Env) of interest are used to deliver the tat gene to TZM-bl cells, which are grown from a CXCR4-positive HeLa cell clone that was engineered to express HIV receptor CD4 and co-receptor CCR5 (Wei et al., 2003Wei X. Decker J.M. Wang S. Hui H. Kappes J.C. Wu X. Salazar-Gonzalez J.F. Salazar M.G. Kilby J.M. Saag M.S. et al.Antibody neutralization and escape by HIV-1.Nature. 2003; 422: 307-312Crossref PubMed Scopus (1647) Google Scholar). During the single-cycle infection that occurs in the TZM-bl cell, the viral Tat protein induces reporter gene expression in trans. Neutralization is scored as the reduction of Tat-regulated firefly luciferase reporter gene expression activity that is quantified as relative luminescence units (RLU) using a luminometer. This type of assay can be performed with large panels of reference strains in multi-well plates to achieve high-throughput capacity (deCamp et al., 2014deCamp A. Hraber P. Bailer R.T. Seaman M.S. Ochsenbauer C. Kappes J. Gottardo R. Edlefsen P. Self S. Tang H. et al.Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies.J. Virol. 2014; 88: 2489-2507Crossref PubMed Scopus (74) Google Scholar, Mascola et al., 2005Mascola J.R. D’Souza P. Gilbert P. Hahn B.H. Haigwood N.L. Morris L. Petropoulos C.J. Polonis V.R. Sarzotti M. Montefiori D.C. Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines.J. Virol. 2005; 79: 10103-10107Crossref PubMed Scopus (195) Google Scholar). The A3R5 cell system is similar, using expression of a Renilla luciferase reporter gene to induce expression by viral Tat protein in cis in a CEM human LCL that naturally expresses the receptor CD4 and co-receptor CXCR4 and that was engineered to express the co-receptor CCR5 (McLinden et al., 2013McLinden R.J. Labranche C.C. Chenine A.L. Polonis V.R. Eller M.A. Wieczorek L. Ochsenbauer C. Kappes J.C. Perfetto S. Montefiori D.C. et al.Detection of HIV-1 neutralizing antibodies in a human CD4+/CXCR4+/CCR5+ T-lymphoblastoid cell assay system.PLoS ONE. 2013; 8: e77756Crossref PubMed Scopus (0) Google Scholar). In this system, engineered viruses that possess a reporter gene in the viral genome are used (Edmonds et al., 2010Edmonds T.G. Ding H. Yuan X. Wei Q. Smith K.S. Conway J.A. Wieczorek L. Brown B. Polonis V. West J.T. et al.Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.Virology. 2010; 408: 1-13Crossref PubMed Scopus (121) Google Scholar). This type of system allows pseudotyping of the reporter particles so that highly pathogenic organisms can be mimicked in biosafety level (BSL) 2 laboratories for agents that are typically restricted for use in BSL3 or BSL4 settings. This approach also allows conventional laboratories to study neutralization of viruses that, if possessed as a live agent, would require select agent registration or even to perform neutralization assays for viruses for which propagation is not permitted. For example, in 2012, when there was a moratorium on use of respiratory droplet-transmissible (rdt) live avian H5N1 influenza virus strains, human mAb-mediated neutralization of these viruses was studied using lentivirus-based pseudovirions packaged with the hemagglutinin (HA) protein of the rdt viruses (Thornburg et al., 2013Thornburg N.J. Nannemann D.P. Blum D.L. Belser J.A. Tumpey T.M. Deshpande S. Fritz G.A. Sapparapu G. Krause J.C. Lee J.H. et al.Human antibodies that neutralize respiratory droplet transmissible H5N1 influenza viruses.J. Clin. Invest. 2013; 123: 4405-4409Crossref PubMed Scopus (18) Google Scholar). More re" @default.
• W2743687550 created "2017-08-17" @default.
• W2743687550 creator A5046971682 @default.
• W2743687550 date "2017-08-01" @default.
• W2743687550 modified "2023-10-16" @default.
• W2743687550 title "Principles of Broad and Potent Antiviral Human Antibodies: Insights for Vaccine Design" @default.
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