Abstract
Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.
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References
Newman L, Rowley J, Hoorn Vander S et al (2015) Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One 10:1–17
Satpathy G, Behera H, Ahmed N (2017) Chlamydial eye infections: current perspectives. Indian J Ophthalmol 65:97
Lijek RS, Helble JD, Olive AJ et al (2018) Pathology after Chlamydia trachomatis infection is driven by nonprotective immune cells that are distinct from protective populations. Proc Natl Acad Sci 115:2216
Nicolle C, Cuenod A, Baizot L (1913) Etude experimentale du trachome. Les Arch l’Institut Pasteur Tunis 4:157–182
Collier LH, Blyth WA (1966) Immunogenicity of experimental trachoma vaccines in baboons. II. Experiments with adjuvants, and tests of cross-protection. J Hyg (Lond) 64:529–544
Olsen AW, Follmann F, Erneholm K et al (2015) Protection against chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein. J Infect Dis 212:978–989
Batteiger BE, Rank RG, Bavoil PM et al (1993) Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis. J Gen Microbiol 139:2965–2972
Badamchi-Zadeh A, McKay PF, Korber BT et al (2016) A multi-component prime-boost vaccination regimen with a consensus MOMP antigen enhances chlamydia trachomatis Clearance. Front Immunol 7:162
Pal S, Peterson EM, de la Maza LM (2005) Vaccination with the chlamydia trachomatis major outer membrane protein can elicit an immune response as protective as that resulting from inoculation with live bacteria. Infect Immun 73:8153–8160
Morrison SG, Su H, Caldwell HD et al (2000) Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4+ T cells but not CD8+ T cells. Infect Immun 68:6979–6987
Morrison RP, Feilzer K, Tumas DB (1995) Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection. Infect Immun 63:4661–4668
Plummer FA, Simonsen JN, Cameron DW et al (1991) Cofactors in male-female sexual transmission of human immunodeficiency virus type 1. J Infect Dis 163:233–239
Morrison SG, Morrison RP (2001) Resolution of secondary Chlamydia trachomatis genital tract infection in immune mice with depletion of both CD4+ and CD8+ T cells. Infect Immun 69:2643–2649
Morrison SG, Morrison RP (2005) A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection. J Immunol 175:7536–7542
Scheid JF, Mouquet H, Feldhahn N et al (2009) A method for identification of HIV gp140 binding memory B cells in human blood. J Immunol Methods 343:65–67
Hayakawa K, Ishii R, Yamasaki K et al (1987) Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells. Proc Natl Acad Sci U S A 84:1379–1383
Doucett VP, Gerhard W, Owler K et al (2005) Enumeration and characterization of virus-specific B cells by multicolor flow cytometry. J Immunol Methods 303:40–52
Woda M, Friberg H, Currier JR et al (2016) Dynamics of dengue virus (DENV)–specific B cells in the response to DENV serotype 1 infections, using flow cytometry with labeled virions. J Infect Dis 214:1001–1009
Degauque N, Ngono AE, Akl A et al (2013) Characterization of antigen-specific B cells using nominal antigen-coated flow-beads. PLoS One 8:e84273
Lugaajju A, Reddy SB, Rönnberg C et al (2015) Novel flow cytometry technique for detection of Plasmodium falciparum specific B-cells in humans: increased levels of specific B-cells in ongoing infection. Malar J 14:370
Frohlich K, Hua Z, Wang J et al (2012) Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria. J Microbiol Methods 91:222–230
Acknowledgments
We would like to thank Prof. Martin Holland and Dr. Harry Pickering of the London School of Hygiene and Tropical Medicine, and Drs. Frank Follmann, Ida Rosenkrands, and Anja Olsen of the Statens Serum Institut for providing the purified elementary bodies used for this work.
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Pinder, C.L., McKay, P.F., Shattock, R.J. (2021). Use of Chlamydial Elementary Bodies as Probes to Isolate Pathogen-Specific Human Monoclonal Antibodies. In: Pfeifer, B.A., Hill, A. (eds) Vaccine Delivery Technology. Methods in Molecular Biology, vol 2183. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0795-4_3
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DOI: https://doi.org/10.1007/978-1-0716-0795-4_3
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