Journal of Microbiology

, Volume 55, Issue 12, pp 973–978 | Cite as

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

  • Kyoung Whun Kim
  • Soyoung Jeong
  • Ki Bum Ahn
  • Jae Seung Yang
  • Cheol-Heui Yun
  • Seung Hyun HanEmail author
Microbial Pathogenesis and Host-Microbe Interaction


The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.


Vibrio cholerae vibriocidal assay guinea pig complement cholera vaccine 


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  1. Ahmed, S., Bardhan, P.K., Iqbal, A., Mazumder, R.N., Khan, A.I., Islam, M.S., Siddique, A.K., and Cravioto, A. 2011. The 2008 cholera epidemic in Zimbabwe: experience of the icddr,b team in the field. J. Health Popul. Nutr. 29, 541–546.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Anh, D.D., Canh, D.G., Lopez, A.L., Thiem, V.D., Long, P.T., Son, N.H., Deen, J., von Seidlein, L., Carbis, R., Han, S.H., et al. 2007. Safety and immunogenicity of a reformulated Vietnamese bivalent killed, whole-cell, oral cholera vaccine in adults. Vaccine 25, 1149–1155.CrossRefPubMedGoogle Scholar
  3. Attridge, S.R., Johansson, C., Trach, D.D., Qadri, F., and Svennerholm, A.M. 2002. Sensitive microplate assay for detection of bactericidal antibodies to Vibrio cholerae O139. Clin. Diagn. Lab. Immunol. 9, 383–387.PubMedPubMedCentralGoogle Scholar
  4. Aung, K.M., Sjostrom, A.E., von Pawel-Rammingen, U., Riesbeck, K., Uhlin, B.E., and Wai, S.N. 2016. Naturally occurring IgG antibodies provide innate protection against Vibrio cholerae bacteremia by recognition of the outer membrane protein U. J. Innate Immun. 8, 269–283.CrossRefPubMedGoogle Scholar
  5. Baumgarten, A. 1980. Viral immunodiagnosis. Yale J. Biol. Med. 53, 71–83.PubMedPubMedCentralGoogle Scholar
  6. Blanco, L.P. and Dirita, V.J. 2006. Antibodies enhance interaction of Vibrio cholerae with intestinal M-like cells. Infect. Immun. 74, 6957–6964.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Boutonnier, A., Dassy, B., Dumenil, R., Guenole, A., Ratsitorahina, M., Migliani, R., and Fournier, J.M. 2003. A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139. J. Microbiol. Methods 55, 745–753.CrossRefPubMedGoogle Scholar
  8. Butler, S.M. and Camilli, A. 2005. Going against the grain: chemotaxis and infection in Vibrio cholerae. Nat. Rev. Microbiol. 3, 611–620.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chen, W.H., Greenberg, R.N., Pasetti, M.F., Livio, S., Lock, M., Gurwith, M., and Levine, M.M. 2014. Safety and immunogenicity of single-dose live oral cholera vaccine strain CVD 103-HgR, prepared from new master and working cell banks. Clin. Vaccine Immunol. 21, 66–73.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chin, C.S., Sorenson, J., Harris, J.B., Robins, W.P., Charles, R.C., Jean-Charles, R.R., Bullard, J., Webster, D.R., Kasarskis, A., Peluso, P., et al. 2011. The origin of the Haitian cholera outbreak strain. N. Engl. J. Med. 364, 33–42.CrossRefPubMedGoogle Scholar
  11. Cohen, I.R. and Norins, L.C. 1968. Antibiodies of the IgG, IgM, and IgA classes in newborn and adult sera reactive with Gram-negative bacteria. J. Clin. Invest. 47, 1053–1062.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Finkelstein, R.A. 1962. Vibriocidal Antibody Inhibition (VAI) analysis: A technique for the identification of the predominant vibriocidal antibodies in serum and for the detection and identification of Vibrio cholerae antigens. J. Immunol. 89, 264–271.Google Scholar
  13. Kanungo, S., Lopez, A.L., Ali, M., Manna, B., Kim, D.R., Mahapatra, T., Holmgren, J., Dhingra, M.S., Weirzba, T.F., Nair, G.B., et al. 2014. Vibriocidal antibody responses to a bivalent killed whole-cell oral cholera vaccine in a phase III trial in Kolkata, India. PLoS One 9, e96499.CrossRefGoogle Scholar
  14. Kanungo, S., Paisley, A., Lopez, A.L., Bhattacharya, M., Manna, B., Kim, D.R., Han, S.H., Attridge, S., Carbis, R., Rao, R., et al. 2009}. Immune responses following one and two doses of the reformulated, bivalent, killed, whole-cell, oral cholera vaccine among adults and children in Kolkata, India: a randomized, placebocontrolled trial. Vaccine 27, 6887–6893.CrossRefPubMedGoogle Scholar
  15. Mahalanabis, D., Lopez, A.L., Sur, D., Deen, J., Manna, B., Kanungo, S., von Seidlein, L., Carbis, R., Han, S.H., Shin, S.H., et al. 2008. A randomized, placebo-controlled trial of the bivalent killed, whole-cell, oral cholera vaccine in adults and children in a cholera endemic area in Kolkata, India. PLoS One 3, e2323.CrossRefGoogle Scholar
  16. Muschel, L.H. and Treffers, H.P. 1956. Quantitative studies on the bactericidal actions of serum and complement. II. Some implications for the mechanism of the bactericidal reaction. J. Immunol. 76, 11–19.PubMedGoogle Scholar
  17. Nguyen, V.D., Sreenivasan, N., Lam, E., Ayers, T., Kargbo, D., Dafae, F., Jambai, A., Alemu, W., Kamara, A., Islam, M.S., et al. 2014. Cholera epidemic associated with consumption of unsafe drinking water and street-vended water-Eastern Freetown, Sierra Leone, 2012. Am. J. Trop. Med. Hyg. 90, 518–523.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Ramamurthy, T., Yamasaki, S., Takeda, Y., and Nair, G.B. 2003. Vibrio cholerae O139 Bengal: odyssey of a fortuitous variant. Microbes Infect. 5, 329–344.CrossRefPubMedGoogle Scholar
  19. Saddler, J.N. and Wardlaw, A.C. 1978. Superiority of human complement for assaying bacterial lipopolysaccharides by their anticomplementary activity. Experientia 34, 1227–1228.CrossRefPubMedGoogle Scholar
  20. Sethi, J., Pei, D., and Hirshaut, Y. 1981. Choice and specificity of complement in complement fixation assay. J. Clin. Microbiol. 13, 888–890.PubMedPubMedCentralGoogle Scholar
  21. Shin, S., Desai, S.N., Sah, B.K., and Clemens, J.D. 2011. Oral vaccines against cholera. Clin. Infect. Dis. 52, 1343–1349.CrossRefPubMedGoogle Scholar
  22. Stroeher, U.H., Karageorgos, L.E., Morona, R., and Manning, P.A. 1992. Serotype conversion in Vibrio cholerae O1. Proc. Natl. Acad. Sci. USA 89, 2566–2570.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Svennerholm, A.M., Jertborn, M., Gothefors, L., Karim, A.M., Sack, D.A., and Holmgren, J. 1984. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J. Infect. Dis. 149, 884–893.CrossRefPubMedGoogle Scholar
  24. Vu, D.T., Hossain, M.M., Nguyen, D.S., Nguyen, T.H., Rao, M.R., Do, G.C., Naficy, A., Nguyen, T.K., Acosta, C.J., Deen, J.L., et al. 2003. Coverage and costs of mass immunization of an oral cholera vaccine in Vietnam. J. Health Popul. Nutr. 21, 304–308.PubMedGoogle Scholar
  25. Weidanz, W.P. and Landy, M. 1963. A simplified method for bactericidal assay of natural antibodies against Gram-negative bacteria. Proc. Soc. Exp. Biol. Med. 113, 861–867.CrossRefPubMedGoogle Scholar
  26. Yang, J.S., Choi, S., Kim, D.D., Kang, S.S., Yun, C.H., Lee, K., and Han, S.H. 2009. A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes. J. Microbiol. Methods 79, 289–294.CrossRefPubMedGoogle Scholar
  27. Yang, J.S., Kim, H.J., Yun, C.H., Kang, S.S., Im, J., Kim, H.S., and Han, S.H. 2007. A semi-automated vibriocidal assay for improved measurement of cholera vaccine-induced immune responses. J. Microbiol. Methods 71, 141–146.CrossRefPubMedGoogle Scholar
  28. Zollinger, W.D. and Mandrell, R.E. 1983. Importance of complement source in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide. Infect. Immun. 40, 257–264.PubMedPubMedCentralGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Kyoung Whun Kim
    • 1
    • 2
    • 3
  • Soyoung Jeong
    • 1
  • Ki Bum Ahn
    • 1
    • 4
  • Jae Seung Yang
    • 3
  • Cheol-Heui Yun
    • 2
    • 5
  • Seung Hyun Han
    • 1
    Email author
  1. 1.Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of DentistrySeoul National UniversitySeoulRepublic of Korea
  2. 2.Department of Agricultural Biotechnology and Research Institute for Agriculture and Life SciencesSeoul National UniversitySeoulRepublic of Korea
  3. 3.Clinical Research Laboratory, Sciences UnitInternational Vaccine InstituteSeoulRepublic of Korea
  4. 4.Research Division for BiotechnologyKorea Atomic Energy Research InstituteJeongeupRepublic of Korea
  5. 5.Institute of Green Bio Science TechnologySeoul National UniversitySeoulRepublic of Korea

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