Parasitology Research

, Volume 108, Issue 4, pp 887–897 | Cite as

Immunogenicity and in vitro protective efficacy of recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa merozoite surface protein-1 (MSP-119) antigen of Plasmodium falciparum

  • Asma Abdullah Nurul
  • Mohd Nor NorazmiEmail author
Original Paper


Vaccine development against the blood-stage malaria parasite is aimed at reducing the pathology of the disease. We constructed a recombinant Mycobacterium bovis bacille Calmette Guerin (rBCG) expressing the 19 kDa C-terminus of Plasmodium falciparum merozoite surface protein-1 (MSP-119) to evaluate its protective ability against merozoite invasion of red blood cells in vitro. A mutated version of MSP-119, previously shown to induce the production of inhibitory but not blocking antibodies, was cloned into a suitable shuttle plasmid and transformed into BCG Japan (designated rBCG016). A native version of the molecule was also cloned into BCG (rBCG026). Recombinant BCG expressing the mutated version of MSP-119 (rBCG016) elicited enhanced specific immune response against the epitope in BALB/c mice as compared to rBCG expressing the native version of the epitope (rBCG026). Sera from rBCG016-immunized mice contained significant levels of specific IgG, especially of the IgG2a subclass, against MSP-119 as determined by enzyme-linked immunosorbent assay. The sera was reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA) and inhibited merozoite invasion of erythrocytes in vitro. Furthermore, lymphocytes from rBCG016-immunized mice demonstrated higher proliferative response against the MSP-119 antigen as compared to those of rBCG026- and BCG-immunized animals. rBCG expressing the mutated version of MSP-119 of P. falciparum induced enhanced humoral and cellular responses against the parasites paving the way for the rational use of rBCG as a blood-stage malaria vaccine candidate.


Malaria Bacille Calmette Guerin Preimmune Seron Merozoite Invasion Falciparum Merozoite 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Anthony Holder for the gifts of 12.10 and 1E1 mAbs and recombinant MSP-119 protein. We are also thankful to Nor Rain Abdullah for the gift of 3D7 P. falciparum. We also thank Rapeah Suppian, Jamaruddin Mat Asan, Munira Grainger, Irene Ling, and Judith Green for their technical assistance. This work was supported by the Malaysian Institute of Pharmaceuticals and Nutraceuticals Special Initiative Grant [No. 304/PPSK/6150077.I121]. NAN was supported by the Malaysian National Science Fellowship (NSF).


  1. Ahlborg N, Ling IT, Holder AA, Riley EM (2000) Linkage of exogenous T-cell epitopes to the 19-kDa region of P. yoelii merozoite surface protein 1 (MSP119) can enhance protective immunity against malaria and modulate the immunoglobulin subclass response to MSP119. Infect Immun 68:2102–2109PubMedCrossRefGoogle Scholar
  2. Aidoo M, Udhayakumar V (2000) Field studies of cytotoxic T lymphocytes in malaria infections: implications for malaria vaccine development. Parasitol Today 16:50–56PubMedCrossRefGoogle Scholar
  3. Aucan C, Traoré Y, Tall F, Nacro B, Traoré-Leroux T, Fumoux F, Rihet P (2000) High immunoglobulin G2 (IgG2) and low IgG4 levels are associated with human resistance to P. falciparum malaria. Infect Immun 68:1252–1258PubMedCrossRefGoogle Scholar
  4. Bergmann-Leitner ES, Duncan EH, Mullen GE, Burge JR, Khan F, Long CA, Angov E, Lyon JA (2006) Critical evaluation of different methods for measuring the functional activity of antibodies against malaria blood stage antigens. Am J Trop Med Hyg 75:437–442PubMedGoogle Scholar
  5. Blackman MJ, Heidrich HG, Donachie S, McBride JS, Holder AA (1990) A single fragment of a malaria merozoite surface protein remains on the parasite during red cell invasion and is the target of invasion-inhibiting antibodies. J Exp Med 172:379–382PubMedCrossRefGoogle Scholar
  6. Bodnar K, Serbina NV, Flynn JL (2001) Fate of M. tuberculosis within murine dendritic cells. Infect Immun 69:800–809PubMedCrossRefGoogle Scholar
  7. Branch OH, Udhayakumar V, Hightower AW, Oloo AJ, Hawley W, Bloland PB, Kaslow DC, Lal AA (1998) A longitudinal investigation of IgG and IgM antibody responses to the merozoite surface protein-1 19 kDa domain of P. falciparum in pregnant women and infants: associations with febrile illness, parasitaemia and anaemia. Am J Trop Med Hyg 58:211–219PubMedGoogle Scholar
  8. Chitarra V, Holm I, Bentley GA, Petres S, Longacre S (1999) The crystal structure of C-terminal merozoite surface protein 1 at 1.8 A resolution, a highly protective malaria vaccine candidate. Mol Cell 3:457–464PubMedCrossRefGoogle Scholar
  9. de Koning-Ward TF, O’Donnell RA, Drew DR, Thomson R, Speed TP, Crabb BS (2003) A new rodent model to assess blood stage immunity to the P. falciparum antigen merozoite surface protein-119 reveals a protective role for invasion inhibitory antibodies. J Exp Med 198:869–875PubMedCrossRefGoogle Scholar
  10. de Souza JB, Ling IT, Ogun SA, Holder AA, Playfair JHL (1996) Cytokines and antibody subclass associated with protective immunity against blood-stage malaria in mice vaccinated with the MSP-1C plus a novel adjuvant. Infect Immun 64:3532–3536PubMedGoogle Scholar
  11. Egan AF, Morris J, Barnish G, Allen S, Greenwood BM, Kaslow DC, Holder AA, Riley EM (1996) Clinical immunity to P. falciparum malaria is associated with serum antibodies to the 19 kDa C-terminal fragment of the merozoite surface antigen, PfMSP1. J Infect Dis 173:765–769PubMedGoogle Scholar
  12. Egan AF, Burghaus P, Druilhe P, Holder AA, Riley EM (1999) Human antibodies to the 19 kDa C-terminal fragment of P. falciparum merozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunol 21:133–139PubMedCrossRefGoogle Scholar
  13. Eisenhut M (2007) Immunity to blood stages of P. falciparum is dependent on a specific pattern of immunoglobulin subclass responses to multiple blood stage antigens. Med Hypotheses 69:804–808PubMedCrossRefGoogle Scholar
  14. Girard MP, Reed ZH, Friede M, Kieny MP (2007) A review of human vaccine research and development: malaria. Vaccine 25:1567–1580PubMedCrossRefGoogle Scholar
  15. Govan VA, Christensen ND, Berkower C, Jacobs WR Jr, Williamson AL (2006) Immunisation with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L1 gene provides protection from CRPV challenge. Vaccine 24:2087–2093PubMedCrossRefGoogle Scholar
  16. Hirunpetcharat C, Tian JH, Kaslow DC, Rooijen NV, Kumar K, Berzofsky JA, Miller LH, Good M (1997) Complete protective immunity induced in mice by immunization with 19 kDa C-terminal fragment of P. yoelii expressed in Saccharomyces cerevisiae. J Immunol 159:3400–3411PubMedGoogle Scholar
  17. Hodder AN, Crewther PE, Matthew ML, Reid GE, Moritz RL, Simpson RJ, Anders RF (1996) The disulfide bond structure of Plasmodium apical membrane antigen-1. J Biol Chem 271:29446–29452PubMedCrossRefGoogle Scholar
  18. Holder AA, Guevara Patiño JA (1997) Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies. J Exp Med 186:1689–1699PubMedCrossRefGoogle Scholar
  19. Holder AA, Guevara Patino JA, Uthaipibull C, Syed SEH, Ling IT, Scott-Finnigan T, Blackman MJ (1999) Merozoite surface protein-1, immune evasion and vaccines against asexual blood stage malaria. Parassitologia 41:409–414PubMedGoogle Scholar
  20. Hui G, Hashimoto C (2007) Plasmodium falciparum anti-MSP1-19 antibodies induced by MSP1-42 and MSP1-19 based vaccines differed in specificity and parasite growth inhibition in terms of recognition of conserved versus variant epitopes. Vaccine 25:948–956PubMedCrossRefGoogle Scholar
  21. Jacobs P, Radzioch D, Stevenson MM (1996) A Th1-associated increase in tumor necrosis factor-α expression in the spleen correlates with resistance to blood-stage malaria in mice. Infect Immun 64:535–541PubMedGoogle Scholar
  22. Jason J, Archibald LK, Nwanyanwu OC, Bell M, Buchanan I, Larned J (2001) Cytokines and malaria parasitemia. Clin Immunol 100:208–218PubMedCrossRefGoogle Scholar
  23. Kaslow DC, Hui G, Kumar S (1994) Expression and antigenicity of Plasmodium falciparum major merozoite surface protein (MSP1(19)) variants secreted from Saccharomyces cerevisiae. Mol Biochem Parasitol 63:283–289PubMedCrossRefGoogle Scholar
  24. Kitua AY, Urassa H, Wechsler M, Smith T, Vounatsou P, Weiss NA, Alonso PL, Tanner M (1999) Antibodies against P. falciparum vaccine candidates in infants in an area of intense and perennial transmission: relationships with clinical malaria and with entomological inoculation rates. Parasite Immunol 21:307–317PubMedCrossRefGoogle Scholar
  25. Lee EAM, Palmer DR, Flanagan KL, Reece WH, Odhiambo K (2002) Induction of T helper type 1 and 2 responses to 19-kilodalton merozoite surface protein 1 in vaccinated healthy volunteers and adults naturally exposed to malaria. Infect Immun 70:1417–1421PubMedCrossRefGoogle Scholar
  26. Long CA, Daly TM, Kima P, Srivastava I (1994) Immunity to erythrocytic stages of malaria parasites. Am J Trop Med Hyg 50(Suppl):27–32PubMedGoogle Scholar
  27. Matsumoto S, Yukitake H, Kanbara H, Yamada T (1998) Recombinant M. bovis BCG secreting merozoite surface protein 1(MSP-1) induces protection against rodent malaria parasite infection depending on MSP-1 stimulated interferon-γ and parasite-specific antibodies. J Exp Med 188:845–854PubMedCrossRefGoogle Scholar
  28. Matsumoto S, Yukitake H, Kanbara H, Yamada T (1999) Long-lasting protective immunity against rodent malaria parasite infection at the blood stage by recombinant BCG secreting merozoite surface protein-1. Vaccine 18:832–834PubMedCrossRefGoogle Scholar
  29. Matsumoto S, Yukitake H, Kanbara H, Yamada H, Kitamura A, Yamada T (2001) M. bovis bacillus Calmette-Guerin induces protective immunity against infection by P. yoelii at blood-stage depending on shifting immunity toward Th1 type and inducing protective IgG2a after the parasite infection. Vaccine 19:779–787CrossRefGoogle Scholar
  30. Mederle I, Bourguin I, Ensergueix D, Badell E, Moniz-Peireira J, Gicquel B, Winter N (2002) Plasmidic versus insertional cloning of heterologous genes in M. bovis BCG: impact on in vivo antigen persistence and immune responses. Infect Immun 70:303–314PubMedCrossRefGoogle Scholar
  31. Morgan WD, Birdsall B, Frenkiel TA, Gradwell MG, Burghaus PA, Syed SEH (1999) Solution structure of an EGF module pair from the P. falciparum merozoite surface protein 1. J Mol Biol 209:113–122CrossRefGoogle Scholar
  32. Near KA, Stowers AW, Jankovic D, Kaslow DC (2002) Improved immunogenicity and efficacy of the recombinant 19-kilodalton merozoite surface protein 1 by the addition of oligodeoxynucleotide and aluminum hydroxide gel in a murine malaria vaccine model. Infect Immun 70:692–701PubMedCrossRefGoogle Scholar
  33. Norazmi MN, Zainuddin ZF, Suppian R, Dale JW (1999) The use of assembly PCR for cloning of a malarial epitope into M. smegmatis—importance of overcoming codon bias. Biotechnol Tech 13:485–489CrossRefGoogle Scholar
  34. Nwuba R, Sodeinde O, Anumudu C, Omosun Y, Odaibo A, Holder AA, Nwagwu M (2002) The human immune response to P. falciparum includes both antibodies that inhibit merozoite surface protein 1 secondary processing and blocking antibodies. Infect Immun 70:5328–5331PubMedCrossRefGoogle Scholar
  35. O’Donnell RA, de Koning-Ward TF, Burt RA (2001) Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria. J Exp Med 193:1403–1412PubMedCrossRefGoogle Scholar
  36. Okech BA, Corran PH, Todd J, Joynson-Hicks A, Uthaipibull C, Egwang TG, Holder AA, Riley EM (2004) Fine specificity of serum antibodies to P. falciparum merozoite surface protein, PfMSP-119, predicts protection from malaria infection and high-density parasitemia. Infect Immun 72:1557–1567PubMedCrossRefGoogle Scholar
  37. Omosun YO, Adoro S, Anumudu CI, Odaibo AB, Uthaipibull C, Holder AA, Nwagwu M, Nwubu RI (2009) Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of P. falciparum. Acta Trop 109:208–212PubMedCrossRefGoogle Scholar
  38. Patino JAG, Holder AA, McBride JS, Blackman MJ (1997) Antibodies that inhibit malaria MSP-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies. J Exp Med 186:1689–1699CrossRefGoogle Scholar
  39. Plebanski M, Hill AVS (2000) The immunology of malaria. Curr Opin Immunol 12:437–441PubMedCrossRefGoogle Scholar
  40. Ramasamy R, Ramasamy M, Yasawardena S (2001) Antibodies and P. falciparum merozoites. Trends Parasitol 17:194–197PubMedCrossRefGoogle Scholar
  41. Rapeah S, Norazmi MN (2006) Immunogenicity of a recombinant M. bovis bacille Calmette–Guerin expressing malarial and tuberculosis epitopes. Vaccine 24:3646–3643PubMedCrossRefGoogle Scholar
  42. Rezende CAF, De Moraes MTB, De Souza Matos DC, Mcintoch D, Armoa GR (2005) Humoral response and genetic stability of recombinant BCG expressing hepatitis B surface antigens. J Virol Methods 125:1–9PubMedCrossRefGoogle Scholar
  43. Rosa DS, Iwai LK, Tzelepis F, Bargieri DY (2006) Immunogenicity of a recombinant protein containing the P. vivax vaccine candidate MSP119 and two human CD4+T-cell epitopes administered to non-human primates (Callithrix jacchus jacchus). Microbes Infect 8:2130–2137PubMedCrossRefGoogle Scholar
  44. Sachdeva S, Ahmad G, Malhotra P, Mukherjee P, Chauhan VS (2004) Comparison of immunogenicities of recombinant P. vivax merozoite surface protein 1 19- and 42-kilodalton fragments expressed in E. coli. Infect Immun 72:5775–5782PubMedCrossRefGoogle Scholar
  45. Sachdeva S, Mohmmed A, Dasaradhi PVN, Crabb BS, Katyal A, Malhotra P, Chauhan VS (2005) Immunogenicity and protective efficacy of E. coli expressed P. falciparum merozoite surface protein-142 using human compatible adjuvants. Vaccine 24:2007–2016PubMedCrossRefGoogle Scholar
  46. Singh S, Miura K, Zhou H, Muratova O, Keegan B, Miles A, Martin LB, Saul AJ, Miller LH, Long CA (2006) Immunity to recombinant P. falciparum merozoite surface protein 1 (MSP1): protection in Aotus nancymai monkeys strongly correlates with anti-MSP1 antibody titer and in vitro parasite-inhibitory activity. Infect Immun 74:4573–4580PubMedCrossRefGoogle Scholar
  47. Smith EC, Taylor-Robinson AW (1999) A role for cytokines in potentiation of malaria vaccines through immunological modulation of blood stage infection. Immunol Rev 171:105–123PubMedCrossRefGoogle Scholar
  48. Sterne JAC, Rodrigues LC, Guedes IN (1998) Does the efficacy of BCG decline with time since vaccination? Int J Tuberc Lung Dis 2:200–207PubMedGoogle Scholar
  49. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF, Snapper SB, Barletta RG, Jacobs WR Jr, Bloom BR (1991) New use of BCG for recombinant vaccines. Nature 351:456–460PubMedCrossRefGoogle Scholar
  50. Taylor-Robinson AW (1995) Regulation of immunity to malaria: valuable lessons learned from murine models. Parasitol Today 11:334–342PubMedCrossRefGoogle Scholar
  51. Taylor-Robinson AW, Phillips RS, Severn A, Moncada S, Liew FY (1993) The role of Thl and Th2 cells in a rodent malaria infection. Science 260:1931–1933PubMedCrossRefGoogle Scholar
  52. Uthaipibull C, Syed SEH, Patino JAG, Ling IT, Holder AA (2001) Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite P. falciparum. J Mol Biol 307:1381–1394PubMedCrossRefGoogle Scholar
  53. von der Weid T, Honarvar N, Langhorne J (1996) Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J Immunol 156:2510–2516PubMedGoogle Scholar
  54. Winkler S, Willheim M, Baier K, Schmid D, Aichelburg A, Graninger W, Kremsner PG (1998) Reciprocal regulation of Th1- and Th2-cytokine-producing T cells during clearance of parasitemia in P. falciparum malaria. Infect Immun 66:6040–6044PubMedGoogle Scholar
  55. Woehlbier U, Epp C, Kauth CW, Lutz R, Long CA, Coulibaly B, Kouyaté B, Arevalo-Herrera M, Herrera S, Bujard H (2006) Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum. Infect Immun 74:1313–1322PubMedCrossRefGoogle Scholar
  56. Wykes M, Good MF (2007) A case for whole-parasite malaria vaccines. Int J Parasitol 37:705–712PubMedCrossRefGoogle Scholar
  57. Zheng CF, Xie PM, Chen YT (2003) Immune response induced by rBCG expressing merozoite surface antigen 2 from P. falciparum. Vaccine 20:914–919CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.School of Dental SciencesUniversiti Sains MalaysiaKubang KerianMalaysia
  2. 2.School of Health SciencesUniversiti Sains MalaysiaKubang KerianMalaysia

Personalised recommendations