Isolation and characterization of human polyreactive pneumococcal polysaccharide antibodies

Rebecca S. Thompson, Noor M. Khaskhely, Kristin R. Malhotra, David J. Leggat, Jason Mosakowski, Sadik Khuder, Gary R. McLean, M. A. Julie Westerink

Department of Medical Microbiology and Immunology, University of Toledo, Toledo, USA.
Department of Medicine, University of Toledo, Toledo, USA.
School of Human Sciences, London Metropolitan University, London, UK.

DOI: 10.4236/oji.2012.23012                

Abstract

Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique K_D values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar K_D values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.

Keywords

B Cell; Pneumococcal Polysaccharide; Polyreactive; Natural; Isotype

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	(1997) Prevention of pneumococcal disease: Recommendations of the Advisory Committee on Immunization Practices (ACIP). Morbidity and Mortality Weekly Report, 46, 1-24. http://www.ncbi.nlm.nih.gov/pubmed/9132580
[2]	Whitney, C.G., Farley, M.M., Hadler, J., Harrison, L.H., Bennett, N.M., Lynfield, R., Reingold, A., Cieslak, P.R., Pilishvili, T., Jackson, D., Facklam, R. R., Jorgensen, J.H. and Schuchat, A. (2003) Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. New England Journal of Medicine, 348, 1737-1746. doi:10.1056/NEJMoa022823
[3]	Avery, O.T.A.W.F.G. (1929) Chemo-immunological studies on conjugated carbohydrate proteins. II. Immuno-logical specificity of synthetic sugar-protein antigens. Journal of Experimental Medicine, 50, 533-550. doi:10.1084/jem.50.4.533
[4]	Alugupalli, K.R. and Gerstein, R.M. (2005) Divide and conquer: Division of labor by B-1 B cells. Immunity, 23, 1-2. doi:10.1016/j.immuni.2005.07.001
[5]	Chen, X., Martin, F., Forbush, K.A., Perlmutter, R.M. and Kearney, J.F. (1997) Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone. International Immunology, 9, 27-41. doi:10.1093/intimm/9.1.27
[6]	Martin, F. and Kearney, J.F. (2000) Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity, 12, 39-49. doi:10.1016/S1074-7613(00)80157-0
[7]	Julien, S., Soulas, P., Garaud, J.C., Martin, T. and Pasquali, J.L. (2002) B cell positive selection by soluble self-antigen. Journal of Immunology, 169, 4198-4204.
[8]	Widhopf, G.F., Brinson, D.C., Kipps, T.J. and Tighe, H. (2004) Transgenic expression of a human polyreactive Ig expressed in chronic lymphocytic leukemia generates memory-type B cells that respond to nonspecific immune activation. Journal of Immunology, 172, 2092-2099.
[9]	Griffin, D.O., Holodick, N.E. and Rothstein, T.L. (2011) Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70. Journal of Experimental Medicine, 208, 67-80. doi:10.1084/jem.20101499
[10]	Nahm, M.G.D. (2002) Training manual for enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (PnPg ELISA). Accessed on 18 April 2011. http://www.vaccine.uab.edu
[11]	Romero-Steiner, S., Libutti, D., Pais, L.B., Dykes, J., Anderson, P., Whitin, J.C., Keyserling, H.L. and Carlone, G.M. (1997) Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells. Clinical and Diagnostic Laboratory Immunology, 4, 415-422.
[12]	Shafer, D.E., Toll, B., Schuman, R.F., Nelson, B.L., Mond, J.J. and Lees, A. (2000) Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in proteinpolysaccha-ride conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides. Vaccine, 18, 1273-1281. doi:10.1016/S0264-410X(99)00370-9
[13]	Weitkamp, J.H., Kallewaard, N., Kusuhara, K., Feigelstock, D., Feng, N., Greenberg, H.B. and Crowe, J.E. Jr. (2003) Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles. Journal of Immunological Methods, 275, 223-237. doi:10.1016/S0022-1759(03)00013-9
[14]	Welschof, M., Terness, P., Kolbinger, F., Zewe, M., Dubel, S., Dorsam, H., Hain, C., Finger, M., Jung, M., Moldenhauer, G., et al. (1995) Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes. Journal of Immunological Methods, 179, 203-214. doi:10.1016/0022-1759(94)00286-6
[15]	McLean, G.R., Nakouzi, A., Casadevall, A. and Green, N.S. (2000) Human and murine immunoglobulin expression vector cassettes. Molecular Immunology, 37, 837-845. doi:10.1016/S0161-5890(00)00101-2
[16]	Wernette, C.M., Frasch, C.E., Madore, D., Carlone, G., Goldblatt, D., Plikaytis, B., Benjamin, W., Quataert, S.A., Hildreth, S., Sikkema, D. J., Kayhty, H., Jonsdottir, I. and Nahm, M.H. (2003) Enzyme-linked immunosorbent assay for quantitation of human antibodies to pneumococcal polysaccharides. Clinical and Diagnostic Laboratory Immunology, 10, 514-519.
[17]	Mian, I.S., Bradwell, A.R. and Olson, A.J. (1991) Structure, function and properties of antibody binding sites. Journal of Molecular Biology, 217, 133-151. doi:10.1016/0022-2836(91)90617-F
[18]	Russell, N.D., Corvalan, J.R., Gallo, M.L., Davis, C.G. and Pirofski, L. (2000) Production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci. Infection and Immunity, 68, 1820-1826. doi:10.1128/IAI.68.4.1820-1826.2000
[19]	Lucas, A.H., Moulton, K.D., Tang, V.R. and Reason, D.C. (2001) Combinatorial library cloning of human antibodies to Streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14, and 23F. Infection and Immunity, 69, 853-864. doi:10.1128/IAI.69.2.853-864.2001
[20]	Zhou, J., Lottenbach, K.R., Barenkamp, S.J., Lucas, A.H. and Reason, D.C. (2002) Recurrent variable region gene usage and somatic mutation in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F. Infection and Immunity, 70, 4083-4091. doi:10.1128/IAI.70.8.4083-4091.2002
[21]	Reason, D.C. and Zhou, J. (2004) Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide. Clinical Immunology, 111, 132-136. doi:10.1016/j.clim.2003.12.004
[22]	Zhou, J., Lottenbach, K.R., Barenkamp, S.J. and Reason, D.C. (2004) Somatic hypermutation and diverse immunoglobulin gene usage in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae Type 6B. Infection and Immunity, 72, 3505-3514. doi:10.1128/IAI.72.6.3505-3514.2004
[23]	Baxendale, H.E., Johnson, M., Stephens, R.C., Yuste, J., Klein, N., Brown, J.S. and Goldblatt, D. (2008) Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease. Clinical & Experimental Immunology, 151, 51-60. doi:10.1111/j.1365-2249.2007.03535.x
[24]	Ochsenbein, A.F. and Zinkernagel, R.M. (2000) Natural antibodies and complement link innate and acquired immunity. Immunology Today, 21, 624-630. doi:10.1016/S0167-5699(00)01754-0
[25]	Casali, P. and Notkins, A.L. (1989) CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire. Immunology Today, 10, 364-368. doi:10.1016/0167-5699(89)90268-5
[26]	Berland, R. and Wortis, H.H. (2002) Origins and functions of B-1 cells with notes on the role of CD5. Annual Review of Immunology, 20, 253-300. doi:10.1146/annurev.immunol.20.100301.064833
[27]	Hardy, R.R. and Hayakawa, K. (2001) B cell development pathways. Annual Review of Immunology, 19, 595-621. doi:10.1146/annurev.immunol.19.1.595
[28]	Mackenzie, L.E., Youinou, P.Y., Hicks, R., Yuksel, B., Mageed, R.A. and Lydyard, P.M. (1991) Auto-and polyreactivity of IgM from CD5+ and CD5? cord blood B cells. Scandinavian Journal of Immunology, 33, 329-335. doi:10.1111/j.1365-3083.1991.tb01778.x
[29]	Baumgarth, N., Herman, O.C., Jager, G.C., Brown, L.E., Herzenberg, L.A. and Chen, J. (2000) B-1 and B-2 cellderived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. Journal of Experimental Medicine, 192, 271-280. doi:10.1084/jem.192.2.271
[30]	Boes, M., Prodeus, A.P., Schmidt, T., Carroll, M.C. and Chen, J. (1998) A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. Journal of Experimental Medicine, 188, 2381-2386. doi:10.1084/jem.188.12.2381
[31]	Briles, D.E., Nahm, M., Schroer, K., Davie, J., Baker, P., Kearney, J. and Barletta, R. (1981) Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae. Journal of Experimental Medicine, 153, 694-705. doi:10.1084/jem.153.3.694
[32]	Ochsenbein, A.F., Fehr, T., Lutz, C., Suter, M., Brombacher, F., Hengartner, H. and Zinkernagel, R.M. (1999) Control of early viral and bacterial distribution and disease by natural antibodies. Science, 286, 2156-2159. doi:10.1126/science.286.5447.2156
[33]	Baxendale, H.E. and Goldblatt, D. (2006) Correlation of molecular characteristics, isotype, and in vitro functional activity of human antipneumococcal monoclonal anti-bodies. Infection and Immunity, 74, 1025-1031. doi:10.1128/IAI.74.2.1025-1031.2006
[34]	Romero-Steiner, S., Musher, D.M., Cetron, M.S., Pais, L.B., Groover, J.E., Fiore, A.E., Plikaytis, B.D. and Carlone, G.M. (1999) Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity [see comments]. Clinical Infectious Diseases, 29, 281-288. doi:10.1086/520200
[35]	Usinger, W.R. and Lucas, A.H. (1999) Avidity as a determinant of the protective efficacy of human anti-bodies to pneumococcal capsular polysaccharides. Infection and Immunity, 67, 2366-2370.
[36]	Sun, Y., Hwang, Y. and Nahm, M.H. (2001) Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B. Infection and Immunity, 69, 336-344. doi:10.1128/IAI.69.1.336-344.2001
[37]	Greenspan, N.S. and Cooper, L.J. (1992) Intermolecular cooperativity: A clue to why mice have IgG3? Immunology Today, 13, 164-168. doi:10.1016/0167-5699(92)90120-V
[38]	Alonso de Velasco, E., Verheul, A.F., van Steijn, A.M., Dekker, H.A., Feldman, R.G., Fernandez, I.M., Kamerling, J.P., Vliegenthart, J.F., Verhoef, J. and Snippe, H. (1994) Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide and native polysaccharide-protein conjugate vaccines: comparison with human anti-polysaccha-ride 23F IgG. Infection and Immunity, 62, 799-808.
[39]	Brezinschek, H.P., Foster, S.J., Dorner, T., Brezinschek, R.I. and Lipsky, P.E. (1998) Pairing of variable heavy and variable kappa chains in individual naive and memory B cells. Journal of Immunology, 160, 4762-4767.
[40]	Bridges, S.L. Jr., Lee, S.K., Johnson, M.L., Lavelle, J.C., Fowler, P.G., Koopman, W.J. and Schroeder, H.W. Jr. (1995) Somatic mutation and CDR3 lengths of immunog-lobulin kappa light chains expressed in patients with rheumatoid arthritis and in normal individuals. Journal of Clinical Investigation, 96, 831-841. doi:10.1172/JCI118129
[41]	Baxendale, H.E., Davis, Z., White, H.N., Spellerberg, M.B., Stevenson, F.K. and Goldblatt, D. (2000) Immuno-genetic analysis of the immune response to pneumococcal polysaccharide. European Journal of Immunology, 30, 1214-1223. doi:10.1002/(SICI)1521-4141(200004)30:4<1214::AID-IMMU1214>3.0.CO;2-D
[42]	Wang, D., Hubbard, J.M. and Kabat, E.A. (1993) Modeling study of antibody combining sites to (alpha 1-6) dextrans. Predictions of the conformational contribution of VL-CDR3 and J kappa segments to groove-type combining sites. Journal of Biological Chemistry, 268, 20584-20589.
[43]	Ichiyoshi, Y. and Casali, P. (1994) Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments. Journal of Experimental Medicine, 180, 885-895. doi:10.1084/jem.180.3.885
[44]	Casali, P. and Schettino, E.W. (1996) Structure and function of natural antibodies. Current Topics in Microbiology and Immunology, 210, 167-179. doi:10.1007/978-3-642-85226-8_17
[45]	Van Esch, W.J., Reparon-Schuijt, C.C., Hamstra, H.J., van Kooten, C., Logtenberg, T., Breedveld, F.C. and Verweij, C.L. (2002) Polyreactivity of human IgG Fcbinding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis. Journal of Autoimmunity, 19, 241-250. doi:10.1006/jaut.2002.0621
[46]	Schreiber, J.R., Barrus, V., Cates, K.L. and Siber, G.R. (1986) Functional characterization of human IgG, IgM, and IgA antibody directed to the capsule of Haemophilus influenzae type b. Journal of Infectious Diseases, 153, 8-16. doi:10.1093/infdis/153.1.8
[47]	Torres, M., Fernandez-Fuentes, N., Fiser, A. and Casadevall, A. (2007) Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity. PLoS One, 2, e1310. doi:10.1371/journal.pone.0001310
[48]	Morelock, M.M., Rothlein, R., Bright, S.M., Robinson, M.K., Graham, E.T., Sabo, J.P., Owens, R., King, D.J., Norris, S.H., Scher, D.S., et al. (1994) Isotype choice for chimeric antibodies affects binding properties. Journal of Biological Chemistry, 269, 13048-13055.
[49]	Torres, M., May, R., Scharff, M.D. and Casadevall, A. (2005) Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype. Journal of Immunology, 174, 2132-2142.
[50]	McCloskey, N., Turner, M.W., Steffner, P., Owens, R. and Goldblatt, D. (1996) Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses. Immunology, 88, 169-173. doi:10.1111/j.1365-2567.1996.tb00001.x
[51]	Liu, H., Chumsae, C., Gaza-Bulseco, G., Hurkmans, K. and Radziejewski, C.H. (2010) Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis. Analytical Chemistry, 82, 5219-5226. doi:10.1021/ac100575n