• View in gallery

    Proliferative response of undepleted, CD4+ T cell-depleted, and CD8+ T cell-depleted cells in BALB/c (a), B10.D2 (b), B10.BR (c), and B10 (d) mice immunized with Plasmodium vivax peptide pools (see Materials and Methods for details). Data are expressed as delta counts per minute (ΔCPM). The background cpm for the results shown here ranged from 91 to 868.

  • View in gallery

    Number of responders by lymphoproliferative response to Plasmodium vivax circumsporozoite protein peptides and antigens in non-exposed individuals (n = 25), in the first malaria episode group (n = 19), and in the multiple episodes group (n = 24). See Materials and Methods for definition of responder. TT = tetanus toxoid; PPD = purified protein derivative of Mycobacterium tuberculosis.

  • View in gallery

    a, Cytotoxic T lymphocyte (CTL) activity in BALB/c mice against pooled Plasmodium vivax peptides and P. falciparum peptide (f97). b, CTL activity in B10.D2 mice against pooled P. vivax peptides and P. falciparum peptide (f97). All assays was performed at a 50:1 effector:target ratio.

  • View in gallery

    Proliferative responses to P30 (a) and cytotoxic T lymphocyte responses (b). In the lymphoproliferative studies, CD4+ and CD8+ T cells were depleted post-immunization but prior to assay.

  • View in gallery

    Cytotoxic T lymphocyte activity against group I Plasmodium vivax circumsporozoite protein peptides in non-exposed- and P. vivax-exposed individuals at different time points. There were 28 subjects in the non-exposed (NE) group, 53 subjects studied at day seven after admission (D7) group, 58 subjects studied at day 28 after admission (D28) group, and 19 subjects studied at follow-up (F) group.

  • View in gallery

    Antibody response to a, Plasmodium vivax classic repeat peptide (v14) and b, P. vivax variant repeat peptide (v30) in non-exposed (NE), single and multiply exposed individuals at a serum dilution of 1:100 at different time points. Data are expressed as net optical density at 405 nm (OD405) (see Materials and Methods). Some symbols represent multiple points (see Results for percent of positive responses for each group). For definitions of other abbreviations, see Figure 5.

  • 1

    Good MF, Doolan DL, 1999. Immune effector mechanisms in malaria. Curr Opin Immunol 11 :412–419.

  • 2

    Tsuji M, Romero P, Nussenzweig RS, Zavala F, 1990. CD4+ cytolytic T cell clone confers protection against murine malaria. J Exp Med 172 :1353–1357.

    • Search Google Scholar
    • Export Citation
  • 3

    Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF, 1988. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc Natl Acad Sci USA 85 :573–576.

    • Search Google Scholar
    • Export Citation
  • 4

    Hoffman SL, Oster CN, Mason C, Beier JC, Sherwood JA, Ballou WP, Mugambi M, Chulay JD, 1989. Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria. J Immunol 142 :1299–1303.

    • Search Google Scholar
    • Export Citation
  • 5

    Doolan DL, Houghten RA, Good MF, 1991. Location of human cytotoxic T cell epitopes within a polymorphic domain of the Plasmodium falciparum circumsporozoite protein.Int Immunol 3 :511–516.

    • Search Google Scholar
    • Export Citation
  • 6

    Malik A, Egan JE, Houghten RA, Sadoff JC, Hoffman SL, 1991. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. Proc Natl Acad Sci USA 88 :3300–3304.

    • Search Google Scholar
    • Export Citation
  • 7

    Dontfraid F, Cochran MA, Pombo D, Knell JD, Quakyi IA, Kumar S, Houghten RA, Berzofsky JA, Miller LH, Good MF, 1988. Human and murine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein: limited immunogenicity of sporozoites and circumsporozoite protein. Mol Biol Med 5 :185–196.

    • Search Google Scholar
    • Export Citation
  • 8

    Good MF, Kumar S, Weiss WR, Miller LH, 1989. T-cell antigenic sites of the malaria circumsporozoite protein. Blood 74 :895–900.

  • 9

    Kumar S, Miller LH, Quakyi IA, Keister DB, Houghten RA, Maloy WL, Moss B, Berzofsky JA, Good MF, 1988. Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum.Nature 334 :258–260.

    • Search Google Scholar
    • Export Citation
  • 10

    Avrameas S, Ternynck T, 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunosorbents. Immunochemistry 6 :53–66.

    • Search Google Scholar
    • Export Citation
  • 11

    Rosenberg R, Wirtz RA, Lanar DE, Sattabongkot J, Hall T, Waters AP, Prasittisuk C, 1989. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax.Science 245 :973–976.

    • Search Google Scholar
    • Export Citation
  • 12

    Franke ED, Lucas CM, Chauca G, Wirtz RA, Hinostroza S, 1992. Antibody response to the circumsporozoite protein of Plasmodium vivax in naturally infected humans. Am J Trop Med Hyg 46 :320–326.

    • Search Google Scholar
    • Export Citation
  • 13

    Park CG, Chwae YJ, Kim JI, Lee JH, Hur GM, Jeon BH, Koh JS, Han JH, Lee SJ, Park JW, Kaslow DC, Strickman D, Roh CS, 2000. Serologic responses of Korean soldiers serving in malaria-endemic areas during a recent outbreak of Plasmodium vivax.Am J Trop Med Hyg 62 :720–725.

    • Search Google Scholar
    • Export Citation
  • 14

    Franke ED, Lucas CM, Roman San E, 1991. Antibody response in humans to the circumsporozoite protein of Plasmodium vivax.Infect Immun 59 :2836–2838.

    • Search Google Scholar
    • Export Citation
  • 15

    Zavala F, Cochrane AH, Naedin EH, Nussenzweig RS, Nussen-zweig V, 1983. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J Exp Med 157 :1947–1957.

    • Search Google Scholar
    • Export Citation
  • 16

    Qari SH, Goldman IF, Povoa MM, Oliveira S, Alpers MP, Lal AA, 1991. Wide distribution of the variant form of the human malaria parasite Plasmodium vivax.J Biol Chem 266 :16297–16300.

    • Search Google Scholar
    • Export Citation
  • 17

    Qari SH, Goldman IF, Povoa MM, di Santi S, Alpers MP, Lal AA, 1992. Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.Mol Biochem Parasitol 55 :105–113.

    • Search Google Scholar
    • Export Citation
  • 18

    Charoenvit Y, Collins WE, Jones TR, Millet P, Yuan L, Campbell GH, Beaudoin RL, Broderson JR, Hoffman SL, 1991. Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. Science 251 :668–671.

    • Search Google Scholar
    • Export Citation
  • 19

    Collins WE, Sullivan JS, Morris CL, Galland GG, Jue DL, Fang S, Wohlhueter R, Reed RC, Yang C, Hunter RL, Lal AA, 1997. Protective immunity induced in squirrel monkeys with a multiple antigen construct against the circumsporozoite protein of Plasmodium vivax.Am J Trop Med Hyg 56 :200–210.

    • Search Google Scholar
    • Export Citation
  • 20

    Thomas BE, Sridevi K, Chopra N, Haq W, Rao DN, 2001. Inducing a cell-mediated immune response against peptides of the Plasmodium vivax circumsporozoite protein. Ann Trop Med Parasitol 95 :573–586.

    • Search Google Scholar
    • Export Citation
  • 21

    Nardin E, Clavijo P, Mons B, van Belkum A, Ponnudurai T, Nussenzweig RS, 1991. T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee. J Immunol 146 :1674–1678.

    • Search Google Scholar
    • Export Citation
  • 22

    Rodrigues MM, Paiva AC, Dutra AP, Yoshida N, Nakaie C, 1991. Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes. Exp Parasitol 72 :271–277.

    • Search Google Scholar
    • Export Citation
  • 23

    Bilsborough J, Carlisle M, Good MF, 1993. Identification of Caucasian CD4 T cell epitopes on the circumsporozoite protein of Plasmodium vivax.J Immunol 151 :890–899.

    • Search Google Scholar
    • Export Citation
  • 24

    Herrera S, Escobar P, de Plata C, Avila GI, Corradin G, Herrera MA, 1992. Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein. J Immunol 148 :3986–3990.

    • Search Google Scholar
    • Export Citation
  • 25

    Zevering Y, Houghten RA, Frazer IH, Good MF, 1990. Major population differences in T cell response to a malaria sporo-zoite vaccine candidate. Int Immunol 2 :945–955.

    • Search Google Scholar
    • Export Citation
  • 26

    Doolan DL, Khamboonruang C, Beck H-P, Houghten RA, Good MF, 1993. Cytotoxic T lymphocyte (CTL) low-responsiveness to the Plasmodium falciparum circumsporozoite protein in naturally-exposed endemic populations: analysis of human CTL response to most known variants. Int Immunol 5 :37–46.

    • Search Google Scholar
    • Export Citation
  • 27

    Blum-Tivouvanziam U, BeghdadiRais C, Roggero MA, Valmori D, Bertholet S, Bron C, Fasel N, Corradin G, 1994. Elicitation of specific cytotoxic T cells by immunization with malaria soluble synthetic polypeptides. J Immunol 153 :4134–4141.

    • Search Google Scholar
    • Export Citation
  • 28

    Houghten RA, 1985. General method for the rapid solid-phase synthesis of large numbers of peptides: Specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA 82 :5131–5135.

    • Search Google Scholar
    • Export Citation
  • 29

    Gotch F, Rothbard J, Howland K, Townsend A, McMichael A, 1987. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326 :881–882.

    • Search Google Scholar
    • Export Citation
  • 30

    Vejbaesya S, Chantangpol R, Longta P, Chandanayingyong D, 1997. HLA class I typing by one-dimensional isoelectric focusing and identification of the new variants in Thai population. Asian Pac J Allergy Immunol 15 :21–27.

    • Search Google Scholar
    • Export Citation
  • 31

    Chiewsilp P, Mongkolsuk T, Sujirachato K, 1997. A*02 in southern Thai Muslims and central Thais. J Med Assoc Thai 80 :S25–S29.

  • 32

    Valmori D, Romero JF, Men Y, Maryanski JL, Romero P, Corradin G, 1994. Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund’s adjuvant (IFA): further enhancement by pre-injection of IFA alone. Eur J Immunol 24 :1458–1462.

    • Search Google Scholar
    • Export Citation
  • 33

    Zevering Y, Khamboonruang C, Rungruengthanakit K, Tungvi-boonchai L, Ruengpipattanapan J, Bathurst I, Barr P, Good MF, 1994. Life-span of human T-cell responses to determinants from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax.Proc Natl Acad Sci USA 91 :6118–6122.

    • Search Google Scholar
    • Export Citation
  • 34

    Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A, 1989. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 19 :2237–2242.

    • Search Google Scholar
    • Export Citation
  • 35

    Weiss WR, Good MF, Hollingdale MR, Miller LH, Berzofsky JA, 1989. Genetic control of immunity to Plasmodium yoelii sporozoites. J Immunol 143 :4263–4266.

    • Search Google Scholar
    • Export Citation
  • 36

    Huang T, Cheng Q, Allan S, Huang Y, 1994. DNA sequencing of circumsporozoite protein genes of Plasmodium vivax from four different countries in west Pacific region: comparative study on the flank sequences. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 12 :85–92.

    • Search Google Scholar
    • Export Citation
  • 37

    Lalvani A, Hurt N, Aidoo M, Kibatala P, Tanner M, Hill AV, 1996. Cytotoxic T lymphocytes to Plasmodium falciparum epitopes in an area of intense and perennial transmission in Tanzania. Eur J Immunol 26 :773.

    • Search Google Scholar
    • Export Citation
  • 38

    Aley SB, Bates MD, Tam JP, Hollingdale MR, 1986. Synthetic peptides from the circumsporozoite protein of Plasmodium falciparum and Plasmodium knowlesi recognize the human hepatoma cell line HepG2-A16 in vitro.J Exp Med 164 :1915–1922.

    • Search Google Scholar
    • Export Citation
  • 39

    Pancake SJ, Holt GD, Mellouk S, Hoffman SL, 1992. Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates. J Cell Biol 117 :1351–1357.

    • Search Google Scholar
    • Export Citation
  • 40

    Rich KA, George FW IV, Law JL, Martin WJ, 1990. Cell-adhesive motif in region II of malarial circumsporozoite protein. Science 249 :1574–1577.

    • Search Google Scholar
    • Export Citation
  • 41

    Rammensee HG, Friede T, Stevanovic S, 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41 :178–228.

  • 42

    Quakyi IA, Currier J, Fell A, Taylor DW, Roberts T, Houghten RA, England RD, Berzofsky JA, Miller LH, Good MF, 1994. Analysis of human T cell clones specific for conserved peptide sequences within malaria proteins. Paucity of clones responsive to intact parasites. J Immunol 153 :2082–2092.

    • Search Google Scholar
    • Export Citation
  • 43

    Zevering Y, Khamboonruang C, Good MF, 1994. Effect of polymorphism of sporozoite antigens on T-cell activation. Res Im-munol 145 :469–476.

    • Search Google Scholar
    • Export Citation
  • 44

    Arevalo-Herrera M, Valencia AZ, Vergara J, Bonelo A, Fleis-chhauer K, Gonzalez JM, Restrepo JC, Lopez JA, Valmori D, Corradin G, Herrera S, 2002. Identification of HLA-A2 restricted CD8(+) T-lymphocyte responses to Plasmodium vivax circumsporozoite protein in individuals naturally exposed to malaria. Parasite Immunol 24 :161–169.

    • Search Google Scholar
    • Export Citation
  • 45

    Zevering Y, Amante F, Smillie A, Currier J, Smith G, Houghten RA, Good MF, 1992. High frequency of malaria-specific T cells in non-exposed humans. Eur J Immunol 22 :689–696.

    • Search Google Scholar
    • Export Citation
  • 46

    Rodrigues M, Nussenzweig RS, Romero P, Zavala F, 1992. The in vivo cytotoxic activity of CD8+ T cell clones correlates with their levels of expression of adhesion molecules. J Exp Med 175 :895–905.

    • Search Google Scholar
    • Export Citation
  • 47

    Braga EM, Carvalho LH, Fontes CJ, Krettli AU, 2002. Low cellular response in vitro among subjects with long-term exposure to malaria transmission in Brazilian endemic areas. Am J Trop Med Hyg 66 :299–303.

    • Search Google Scholar
    • Export Citation
  • 48

    Goonewardene R, Carter R, Gamage CP, Del Giudice G, David PH, Howie S, Kendis KN, 1990. Human T cell proliferative responses to Plasmodium vivax antigens: evidence of immunosuppression following prolonged exposure to endemic malaria. Eur J Immunol 20 :1387–1391.

    • Search Google Scholar
    • Export Citation
  • 49

    Ho M, Webster HK, Looareesuwan S, Supanaranond W, Phillips RE, Chanthavanich P, Warrell DA, 1986. Antigen-specific immunosuppression in human malaria due to Plasmodium falciparum.J Infect Dis 153 :763–771.

    • Search Google Scholar
    • Export Citation
  • 50

    Troye-Blomberg M, Romero P, Patarroyo ME, Bjorkman A, Perlmann P, 1984. Regulation of the immune response in Plasmodium falciparum malaria. III. Proliferative response to antigen in vitro and subset composition of T cells from patients with acute infection or from immune donors. Clin Exp Immunol 58 :380–387.

    • Search Google Scholar
    • Export Citation
  • 51

    Hirunpetcharat C, Good MF, 1998. Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice following challenge with homologous parasite. Proc Natl Acad Sci USA 95 :1715–1720.

    • Search Google Scholar
    • Export Citation
  • 52

    Wipasa J, Xu H, Stowers A, Good MF, 2001. Apoptotic deletion of helper T cells specific for the 19 kDa carboxyl terminal fragment of merozoite surface protein 1 (MSP119) during malaria infection.J Immunol 167 :3903–3909.

    • Search Google Scholar
    • Export Citation
  • 53

    Xu H, Wipasa J, Yan H, Zeng M, Makobongo MO, Finkelman FD, Kelso A, Good MF, 2002. The mechanism and significance of deletion of parasite-specific CD4+ T cells in malaria infection. J Exp Med 195 :881–892.

    • Search Google Scholar
    • Export Citation
  • 54

    Toure-Balde A, Sarthou JL, Aribot G, Michel P, Trape JR, Ro-gier C, Roussilhon C, 1996. Plasmodium falciparum induces apoptosis in human mononuclear cells. Infect Immun 64 :744–750.

    • Search Google Scholar
    • Export Citation
  • 55

    Gould K, Cossins J, Bastin J, Brownlee GG, Townsend A, 1989. A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. J Exp Med 170 :1051–1056.

    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 283 60 2
PDF Downloads 27 16 2
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

ANALYSIS OF CIRCUMSPOROZOITE PROTEIN–SPECIFIC IMMUNE RESPONSES FOLLOWING RECENT INFECTION WITH PLASMODIUM VIVAX

View More View Less
  • 1 The Cooperative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, Herston, Queensland, Australia; The Tropical Medicine Hospital, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

CD8+ and CD4+ T cells are involved in immunity to the pre-erythrocytic stage of malaria. This study has been undertaken to define T cell epitopes on the Plasmodium vivax circumsporozoite protein (CSP) and to analyze the early induction of immune response following infection. We identified CD4+ and CD8+ T epitopes recognized by different strains of mice as well as by humans. The CD4+ T cell response in mice was found to be similar in all strains, but variation between strains was evident. Five H-2d-restricted CD8+ cytotoxic T lymphocyte (CTL) epitopes, but no H-2k-or H-2b-restricted epitopes, could be defined. Non-H-2 genes were also able to regulate the response. In recently infected Thai adults, poor immunoresponsiveness was demonstrated. CTL activity and proliferative responses of T cells from malaria-exposed donors were very low. In contrast, exposed individuals had specific antibodies against the immunodominant repeats of both common strains of the P. vivax CSP; however, titers decreased following treatment.

INTRODUCTION

Immunity to malaria sporozoites is believed to be mediated by both T cells and antibodies1 and evidence from both rodent2,3 and human studies4 suggests that the circumsporozoite protein (CSP) can be a target of both T cells and antibodies, thus rendering it a leading sporozoite/liver stage vaccine candidate. Data from murine studies have suggested that CD8+ as well as CD4+ T cells are required for protection against malaria,3 and humans who were immunized with irradiated sporozoites of Plasmodium falciparum, as well as those naturally exposed to P. falciparum sporozoites, developed CD8+ and CD4+ T cell responses against the P. falciparum CSP, although such responses were often infrequently present.5,6 Although logistically very difficult, one way to define human CD8+ T cell epitopes on the CSP of P. vivax is to expose volunteers to the bites of hundreds of irradiated mosquitoes heavily infected with P. vivax sporozoites. However, a number of studies have now demonstrated that the location of epitopes on a given protein recognized by human T cells may be very similar to, often overlapping, the region recognized by murine T cells. For example, human and murine CD4+ T cell epitopes map to the same region of the P. falciparum CSP7,8 and a murine CD8+ T cell epitope on the P. falciparum CSP overlaps a human CD8+ T cell epitope.5,6,9

Analysis of antibody responses to P. vivax CSP is important but is complicated by the fact that there are two distinct types of repeats on the P. vivax CSP.10,11 Antibodies against repeats have been reported to occur in the serum of malaria-exposed subjects,12,13 and antibody to non-repeat regions has also been found,14 a situation different to that for P. falciparum, where the vast majority of antibody to sporozoites is directed against the repeats only.15 Since it is believed that CSP-specific CD4+ and CD8+ T cell responses, along with CSP-specific antibodies, will be important in immunity to P. vivax sporozoite/liver stages, and because polymorphisms within the P. vivax CSP16,17 could render a single recombinant CSP ineffective as a vaccine candidate, a major strategy is to define T and B cell epitopes and combine these in a synthetic vaccine. An epitope on P. vivax CSP recognized by a protective monoclonal antibody has been defined,18 and vaccine constructs based on this19 and CSP T cell epitopes20 have been made. Other epitopes defined from monkey21 and murine20,22 studies may also prove useful.

In Thailand, malaria is a significant problem and P. vivax accounts for nearly 30% of all infections. The incidence of P. vivax malaria is higher during the wet season (May-August). It affects mostly non-immune individuals traveling for work to the regions of high endemicity on the borders of Myanmar, Laos, and Cambodia. Although there have been some studies of human immune responses to the P. vivax CSP,12,23–25 these have been limited and only two former studies have looked at T cell responses among Thais.25,26 There have been no studies to define Thai CD8+ T cell epitopes on the CSP of P. vivax. The goals of this study were to facilitate vaccine design and development by analyzing the T and B cell responses specific to P. vivax CSP of recently infected Thais, gain insight into the early induction of CSP-specific immunity, and define the possible location of other important epitopes by examining the immune responses of vaccinated mice.

MATERIALS AND METHODS

Patients.

One hundred thirteen P. vivax-exposed donors (age range = 14-65 years) were recruited from patients from the Kanchanaburi area admitted to The Tropical Medicine Hospital at Mahidol University in Bangkok, Thailand. All exposed donors were slide positive for P. vivax at the time of admission. Some patients had multiple (2–7) episodes of P. vivax infection. The patients were bled on days 0, 7, and 28 after admission. Some follow-up patients were tested one month to two years after their last episode. Thirty-six non-exposed volunteers (age range = 22−55 years) were Bangkok residents with no history of malaria. This research was reviewed and approved by the Bancroft Human Ethics Research Committee. Peripheral blood mononuclear leukocytes were prepared from heparinized blood and tested for their proliferative response and their ability to generate cytotoxic responses in vitro to P. vivax peptides. Serum or plasma was also collected at each time point, as available, for estimation of antibody responses to P. vivax peptides.

Animals.

Six to eight week-old female BALB/c (H-2d), B10.D2 (H-2d), B10.BR (H-2k), B10 (H-2b), and F1 BALB/c × B10.D2 mice were obtained from the Animal Resources Center, Willetton, Western Australia.

Peptides and antigens.

20-mer overlapping peptides spanning the entire sequence of P. vivax (Belem and North Korean strains and the repeat variants of the VK strain), a CD8+ epitope of P. falciparum CSP, f97 (IEKYLKTIKNSL-STEWSPCS) recognized by BALB/c mice,27 and f57 (KP-KDELDYENDIEKKICKMEKCS), a CD8+ epitope from P. falciparum CSP recognized by B10.BR mice9 were used. These peptides were synthesized by the tea bag method28 at The Queensland Institute of Medical Research and their sequences are given (Table 1). Because of the large number of peptides to be tested and the difficult logistics of testing peripheral blood mononuclear leukocytes to each peptide from only a small volume of blood, peptides were frequently pooled, following a similar methodology in other studies.5 It is possible that by pooling peptides antigenic competition may result in some peptides being non-immunogenic. Major histocompatibility complex (MHC)-linked and non-MHC-linked genes could influence the outcome in a manner partly dependent on the conditions used in culture and assay. For the CD4+ T cell studies in mice, pool A consisted of v1–v10, pool B consisted of v11–v20, and pool C consisted of v21–v30. For the CD8+ T cell studies in mice, group I consisted of v1–v5, group II consisted of v6–v10, group III consisted of v11–v15, group IV consisted of v16–v20, and group V consisted of v21–v26. There were equal amounts of each peptide in the pool. When used for generation of in vitro human cytotoxic T lymphocyte (CTL) cultures, they were grouped as followed: group I (v1–v5), group II (v6–v10), group III (v11–v15 and v27–v30), group IV (v16–v20), and group V (v21–v26). The influenza matrix peptide (residues 55–73, LTKGILGFVF TLTVPSERG-amide) was used as a positive control for human CTL assays. The CTL response to the influenza matrix peptide has previously been described to be restricted by HLA-A2.1.29 The frequency of HLA-A2 in the Thai population is approximately 10%.30 Another report has indicated that the frequency of the HLA-A2.1 subtype is rare.31 Tetanus toxoid (TT) (0.3 flocculation units/mL) (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) and purified protein derivative (PPD) of Mycobacterium tuberculosis (Commonwealth Serum Laboratories) (60 μg/ mL) were used as positive control antigens. Bovine serum albumin (BSA)-coupled peptides were used as antigens in an enzyme-linked immunosorbent assay (ELISA) for the determination of P. vivax-specific antibodies. Peptides were coupled to BSA using the glutaraldehyde coupling method as described.10

Mapping of CD4+ T cell epitopes.

Three to four mice were immunized with 30 μg per animal of pooled peptides emulsified in Freund’s complete adjuvant in the foot pad unless otherwise specified (50 μL per footpad). Inguinal and popliteal lymph nodes were collected 8–9 days after immunization and tested for their proliferative response to P. vivax CSP peptides at concentrations of 30 μg/mL and 10 μg/mL in a volume of 200 μL. Responses were expressed as Δ counts per minute (mean cpm of wells with peptide - mean cpm of wells without peptide). In human studies, lymphocyte proliferation assays were performed against individual peptides at a concentration of 30 μg/mL. Results are expressed as stimulation indices (SIs), where an SI > 3.0 was arbitrarily considered a positive response. Although this is an arbitrary cut-off value, it has been commonly used in similar studies.23,25

Mapping of CD8+ T cell epitopes.

Animals were pre-primed with phosphate-buffered saline emulsified in Freund’s incomplete adjuvant at the tail base three weeks prior to immunizing with 50 μg of peptide also emulsified in Freund’s incomplete adjuvant following an established protocol.32 In vitro CTLs were generated against the appropriate peptide pool at a concentration 10 μg/mL with 30 units/mL of recombinant interleukin-2 (rIL-2) being added on day 2. The culture was tested for CTL activity after 7-9 days. Some cultures were also restimulated with 10 μg/mL of antigen, 30 units/mL of rIL-2, and irradiated spleen cells every two weeks. The conventional chromium release assay was performed 7–8 days after restimulation. P-815 cells were used as target cells for BALB/c and B10.D2 CTL, while L-929 and EL-4 cells were used as targets for B10.BR and B10 cultured cells, respectively.

Human CTL cultures were set up as described5 in 24-well plates. For in vitro secondary restimulation, cultures were fed with autologous mitomycin C-treated phytohemagglutinin-stimulated blasts in the presence of peptides (10 μg/mL) and rIL-2 (30 units/ml) in 25-cm2 flasks. The influenza matrix peptide was used as a positive control (10 μg/mL). The CTL activity was assessed seven days after restimulation. The specific CTL activities were calculated and specific lysis greater than 15% was arbitrarily considered positive, as described elsewhere.5

CD4+ or CD8+ T cell depletion of LN cells or CTL lines.

The LN or CTL cultured cells were incubated with excess amounts of rat anti-mouse CD4 (GK1.5) or CD8 (TIB105) monoclonal antibodies. The depleted cell population was then tested for its T cell activities. The CD4+ T cell depletion ranged from 83% to 97% and CD8+ T cell depletion ranged from 91% to 96% as determined by fluorescence-activated cell sorting analysis.

Detection of P. vivax peptide-specific antibody by ELISA.

Human IgG antibodies against peptides representing the two types of P. vivax CSP repeats (v14 and v30 representing the classic repeats and the VK 247 repeats, respectively) (Table 1) were detected by an ELISA. Peptide was coupled to BSA and then coated onto the ELISA plate at a concentration of 2 μg/mL in coating buffer. Samples where the net optical density at 405 nm (OD405) at a dilution of 1:100 was greater than the mean + 3 SD of the non-exposed samples tested against each peptide were considered positive. In cases where there was a high BSA-specific antibody response that sample was omitted.

RESULTS

CD4+ T cell responses.

Different strains of mice (BALB/c [H-2d], B10.D2 [H-2d], B10.BR [H-2k], and B10 [H-2b]) were immunized with the different pools of peptides as described earlier in this report. Draining LN cells were then cultured with individual peptides to define CD4+ T cell epitopes. For some LN cell populations, cells were depleted prior to culture using antibodies to CD4 or CD8. The CD4+ T cell epitopes were found to be clustered in two regions of the P. vivax CSP, one at the amino terminal region in the first 60 amino acids and the other at a carboxyl terminal region covering a conserved region (region II, comprising amino acids TEWTPCSVTCGVG within peptide v21) of the protein. All four mouse strains responded well to v21 (amino acids 309–328). BALB/c and B10.D2 mice showed the same pattern of responses to all peptides (Figure 1a and b). B10.BR mice responded to the classic repeat peptide (v14) (Figure 1c), while B10 mice responded to the variant repeat peptide (v30) (Figure 1d). The CD4+ T cell depletion prior to in vitro stimulation abrogated most proliferative responses, while the effect on the proliferative response of CD8+ T cell depletion was much less (Figure 1).

Individual humans were also tested for their lymphoproliferative responses (Figure 2). Control antigens (TT and PPD) were included in the study. Both non-exposed and exposed subjects responded at approximately equivalent levels to these control antigens (96% of non-exposed subjects and 98% of exposed subjects responded to PPD, while 63% of non-exposed and 44% of exposed subjects responded to TT). The P. vivax-exposed group was recently exposed and were divided into those with a single exposure and those with multiple exposures. The data are expressed as the percent of responders in a given group of subjects. As mentioned earlier, positivity was defined as a stimulation index greater than 3. Little responsiveness by peripheral blood mononuclear cells of non-exposed subjects to the CSP peptides was found, with one (4%) of 25 responding to each of v6, v7, v9, v20, and v22, and two (8%) of 25 responding to v18. These peptides have been reported to stimulate cells from non-exposed subjects in other studies.23,33 However, malaria-exposed subjects also responded infrequently to the peptides. In those individuals who had their first episode, three (16%) of 19 responded to v2, one (5%) of 19 to v5, one (5%) of 19 to v9, v12, v22, v23, v27, and two (11%) of 19 to v19 (Figure 2). In those individuals with multiple episodes of P. vivax malaria, two (8%) of 24 responded to v1 and v2, one (4%) of 24 to v19, v21, and v25, and three (13%) of 24 to v22 (Figure 2). Levels of responsiveness to CSP peptides by both exposed and non-exposed subjects were much less than that previously observed33 and may reflect the fact that the individuals in this study were recently infected, compared with the other studies.

CD8+ T cell responses.

Using an established protocol (see Materials and Methods) different strains of mice were immunized with the different peptide pools for definition of putative CTL epitopes. BALB/c (H-2d) mice were found to recognize peptides v1, v2, v5, v19, v21, and v25, while B10.D2 mice (also H-2d) repeatedly recognized only v19 (Figure 3). Peptides 21 and 25 did not always induce a CTL response in BALB/c mice, but did so on two of four occasions. The discrepancy in CTL responsiveness between the H-2-identical strains (BALB/c and B10.D2) suggested that there may be negative regulatory non-MHC elements in B10.D2 mice affecting the responsiveness to these epitopes. To assess this possibility, BALB/c and B10.D2 mice were crossed to produce F1 mice that were subsequently tested for immunologic responsiveness. However, the F1 mice were found to recognize the same P. vivax peptides as the parental BALB/c strain, suggesting that the lack of responsiveness was not due to a dominant suppressive regulatory effect. A known P. falciparum H-2d-restricted epitope, f97 (sequence given in the Materials and Methods),27 was also tested in the three H-2d strains and was shown to induce CTL in BALB/c, B10.D2 (Figure 3), and F1 mice. All defined epitopes were shown to be recognized by CD8+ T cells because CTL activity was reduced after CD8 depletion by more than 75%. No CTL activity against P. vivax CSP peptides was demonstrated by either B10 (H-2b) or B10.BR (H-2k) mice using this protocol. A positive control peptide for B10.BR mice (the P. falciparum CSP peptide f57 (sequence given in the Materials and Methods)9 was tested and shown to be immunogenic for CTL activity.

It was of interest that no P. vivax CSP-specific epitopes were defined in B10 or B10.BR mice. A particular concern was the possibility that the lack of response was the result of a lack of suitable CD4+ T cell-mediated help for the generation of CTL. The CD4+ T cell epitopes have been defined on the P. vivax CSP and it was noted that there was a paucity of CD4+ T cell responsiveness among pools II and III. We thus immunized B10 and B10.BR mice with pool II or pool III peptides together with either v21 (well recognized by CD4+ T cells from all strains) or the CD4+ T cell promiscuous epitope from tetanus toxin P30,34 which was confirmed here to be immunogenic in BALB/c, B10.D2, and B10 mice (Figure 4a). Using peptide 21 as a putative source of help for CTL generation, we showed that there was still no generation of CTL activity from B10.BR and B10 mice in response to pools II or III. However, when P30 was used as a source of putative help in B10 mice, CTL were generated to the group II pool containing P30 (pool III was not tested with P30). Analysis of the response, however, showed that the CTL were specific for P30 itself, not the peptides in pool II (Figure 4b). P30 and v21 were similarly unsuccessful in inducing a CTL response to peptides from either pool II or pool III in B10.BR mice. This same approach was then used to assess whether further epitopes could be defined in BALB/c and B10.D2 mice from among the negative pools. No further CTL epitopes from the P. vivax CSP were defined for these mice. However, it was observed that P30 itself was also seen by CD8+ T cells from BALB/c mice (Figure 4b). Curiously, B10.D2 mice failed to recognize this peptide. Presumably, non-MHC genes are contributing to the regulation of this response as has been shown in other systems.35

A polymorphism occurs naturally in peptide v19, the epitope recognized by CD8+ CTL from BALB/c and B10.D2 mice. A single amino acid mutation occurs in the Chinese strain36 at position 300, resulting in a change of amino acid from glutamic acid to valine. A peptide representing the Chinese strain polymorphism (v19var) was thus constructed to determine whether this change affected CTL recognition. For BALB/c and B10.D2 mice, v19-specific CTL (induced following immunization with group IV) completely failed to recognize v19var.

CTL activity against P. vivax peptides in recently exposed and non-exposed Thais.

Peptide-specific responses to all P. vivax CSP peptides tested as pools were low. For the majority of individuals studied, the net peptide-specific lysis was between -10% and +10%, as previously shown in a study of P. falciparum-specific CTL.23 For the group I peptides (Figure 5), CTL from four of 53 individuals tested were greater than 15% (peptide-specific lysis). One individual responded to group V peptides. There were no responses to any other groups. While these may represent genuine peptide-specific lytic activity, it was not possible to re-test these individuals, who had left hospital and returned to their homes (all outside Bangkok) by the time the results of the study were at hand. While the positive results could not be confirmed, it is clear that the frequency of CTL to P. vivax CSP at a population level is very low. This low frequency is similar to the very low frequency of P. falciparum CSP-specific CTL observed in Caucasians, northwestern Thais, Kenyans, Gambian,37 and Papua New Guineans.26

The CTL responses to the influenza matrix peptide were generated in eight of 74 individuals. Although it was not possible to retest individuals, the data do suggest that at the population level there was a low but finite frequency of influenza peptide-specific CTL. Although the influenza peptide-specific response is low, it is similar to the low frequency of HLA.A2.1 individuals in the Thai population,30 and may also represent a lack of recent exposure to influenza. The ability to generate CTL using this methodology both here and previously5,26 give us certainty that our methodology is working.

Antibody response.

Sera of P. vivax-exposed and non-exposed subjects were tested for the presence of antibodies to the “classic” and “variant” repeats of the P. vivax CSP (v14 and v30). The average OD405 + 3 SD of 34 non-exposed sera was used as a cut-off value. None of the non-exposed subjects had antibodies to either v14 or v30. However, unlike the paucity of T cell responsiveness to P. vivax CSP peptides, IgG-specific antibody responses among P. vivax-exposed subjects to the CSP were significant. The classic P. vivax repeat peptide (v14) was recognized by 63% (12 of 19) of the sera from individuals having their first episode when tested on admission, 68% (17 of 25) when tested on day 7, and 33% (8 of 24) when tested on day 28 (Figure 6a). In multiply exposed individuals 50% (14 of 28) were positive on admission, 50% (15 of 30) on day 7, and 18% (6 of 34) on day 28.

The IgG-specific antibody response to the variant P. vivax repeat peptide (v30) was 42% (8 of 19) in first exposed individuals on admission, 52% (13 of 25) at day 7, and 29% (7 of 24) at day 28 (Figure 6b). In multiply exposed individuals 60% (17 of 28) were positive at day 0, 53% (16 of 30) at day 7, and 50% (17 of 34) at day 28.

DISCUSSION

This study defines CSP-specific immune responses in individuals recently infected with P. vivax. Using an overlapping set of synthetic 20-mer peptides, CD4+ and CD8+ T cell epitopes were initially defined for different strains of mice. LN cells from all strains of mice gave a very similar response to peptides spanning two areas of the P. vivax CSP. The first area is amino terminal to the conserved region (region I [RI]). This region, particularly within the first 60 amino acids, is polymorphic, with the polymorphism restricted to amino acid positions 11, 13, 38, 49, and 52.17 The second area is carboxyl terminal to the repeat region, in the vicinity of another conserved region (region II [RII]). Again, this area is also polymorphic.16,17 One peptide (v21) was identified that was recognized by CD4+ T cells from all strains of mice studied. The data concurred with a previous report identifying a peptide with an overlapping sequence as being immunogenic in different mouse strains.22 From available sequence data, this peptide (v21) has only one amino acid variation, V/A at position 322.17 This peptide also includes the conserved region RII, which is thought to be important in hepatocyte invasion.38–40 While not recognized by acutely/very recently infected Thais, v21 is known to be recognized by up to 40% of Caucasians previously exposed to P. vivax.23 It may have particular merit in a P. vivax CSP vaccine by increasing the chance that immunity will be boosted by natural infection. Further investigation of the v21 variant is required.

For murine CD8+ T cells, the response was restricted to one H-2 haplotype (H-2d). Since CTL of BALB/c mice responded to more epitopes than B10.D2 mice and both strains are of the H-2d haplotype, it is likely that non-H-2 genes are contributing to immunologic responsiveness. Similar observations have been previously reported.35 The CTL responses in BALB/c × B10.D2 F1 mice were found to be similar to those in BALB/c mice. This suggests that dominant negative regulatory elements on the B10 background are not responsible for the lack of responsiveness of B10.D2 mice. It is also likely that non-HLA genes may be contributing to the human low CTL immunologic responsiveness to the CSP. The CD8+ T cell epitopes can often be predicted from motifs. Although this approach was not used here to identify epitopes, an analysis of the identified epitopes with respect to motifs has been undertaken. A class I anchor for H-2KdDd cannot be defined on v19 peptide, compared with MHC ligands and peptide motifs listed.41 However, one possible motif on the v19 peptide was identified for H-2Ld (APNEKSVKVYL, where anchor residues are P at position 2 and L at position 11).

Possible MHC class I motifs on v1 and v2 were identified in this study. The preferred H-2Kd anchor residue at position 2 is F and that at position 9 is I.41 Peptide v1 has F at position 2 and I at position 10. Possible anchor residues on v2 are F at position 2 and L at position 12 (LFPTHCGHNVDL). Peptide 5 in group I pool elicited some limited CTL activity and a possible motif could not be identified. A possible class I motif on v21 could not be identified. For v25, possible H-2Kd motif anchor residues are F at position 2 and L at position 9.

Peptide v19 has three known amino acid polymorphisms, P/T (proline to threonine), E/V (glutamic acid to valine), and K/I (lysine to isoleucine),17 and one of these polymorphisms (E/V) was found to completely abrogate CTL recognition. Cross-recognition between the variants requires further study; however, it is tempting to consider that polymorphisms have been selected by P. vivax CSP-specific CTL, as has been postulated to occur with P. falciparum CSP.8 Clearly, however, further consideration of this hypothesis requires confirmation that human CTL recognize v19. Human proliferative T cells (type undefined) have been shown to commonly recognize v19,23 although the ability of the polymorphisms to ablate recognition of human T cells has not been determined.

It was of interest that P30 (from TT, 947-967, FNNFTVS-FWLRVPKVSASHLE), as well as functioning as a promiscuous helper epitope (as previously described34), was also seen as a cytotoxic T epitope in both H-2d and H-2b mice. P30 contains possible anchor residues for defined motifs in both strains. There are two possible sets of H-2Kd anchors (NFTVSFWL, and SFWLRVPKV) and one H-2Kb anchor (FTVSFWLRV).41

The reasons for the low CD8+ responsiveness are unknown. However, we have previously shown that the frequency of CTL responses to epitopes of the P. falciparum CSP was very low among visitors to and residents of endemic areas, and that recent exposure to sporozoites (within two years) was a prerequisite for detection of specific CTL.5 A study by Malik and others6 demonstrated that P. falciparum CSP-specific CTL were detected in four of five recently and heavily exposed volunteers. Given that the subjects in the study presented here were not heavily exposed, it appears that in general, recent and heavy exposure of humans may be required if CSP-specific CTL are to be present in sufficiently high frequency to be detected in the in vitro assays used in this and the previous studies. Other factors referred to in different studies42,43 have also been postulated to affect CSP-specific T cell responsiveness, including paucity of epitopes within the CSP and polymorphism within epitopes resulting in limited exposure to any particular epitope.

In a recent Colombian study,44 three HLA 0201-restricted CD8+ epitopes were defined on the P. vivax CSP. These were located in the carboxyl terminal region of the protein corresponding to residues 301-309, 365-374, and 341-349, closely overlapping some of the regions recognized by the murine CD8+ T cells in our study. Although our human volunteers did not recognize these epitopes, this may relate to the fact that the patients in our study were recently infected and parasitemic at the time of our study, whereas the Columbian volunteers were not, but had 20 years of permanent exposure. The use of tetramers could enable a more detailed analysis of the basis for poor CD8 T cell responsiveness.

The low level of human lymphoproliferative responsiveness was unexpected. We previously showed23 that both malaria-exposed and non-exposed Caucasians responded to multiple epitopes within the P. vivax CSP, but that the exposed donors responded uniquely to a subset of epitopes. We also found that responses were more frequently found in those individuals more recently exposed. In a study in Karen Thais (an ethnic population different from the Thais in this study33), we also demonstrated significantly more responsiveness using similar methodology to that used here. An important difference, however, between the previous studies and the study reported here was that the two previous studies did not examine responsiveness in acutely and very recently infected subjects. Given that the same peptide sequences were studied and that the methodologies of all three studies were very similar, it appears that peptide-specific CD4+ T cells are not present in the peripheral blood of individuals acutely infected in numbers sufficient to detect in the assay used here. Furthermore, since both previous studies demonstrated that responses were found in non-exposed donors (postulated to be the result of immunologic cross-reactivity between CSP and environmental organisms),45 as well as exposed donors, the lack of responsiveness observed here is likely not to be due to insufficient time since exposure to generate a response. This hypothesis is supported by the observation that P. vivax CSP-specific antibodies were readily detected in a number of individuals tested. A likely explanation for the data observed here is that CSP-specific T cells are present in a location other than the peripheral blood. An obvious site would be the liver. In the murine model, CSP-specific protective CTL have been shown to home to the liver.46 It is worth noting that in a recent Brazilian study proliferative responses to recombinant CSP were observed in 50% of the adults not continuously exposed to malaria, but in only 10% of subjects from a transmission area.47 As in our study, antibody responses were similar in both groups. These studies are very complementary and point to the likely effect of recent infection on immunomodu-lation/lymphocyte trafficking, and also highlighted the importance of timing of blood collection when analyzing T cell responses. Many of the factors postulated to be relevant to human T cell non responsiveness (such as polymorphism, suppression, and sequestering of T cells in the liver) would not be operative in mice following peptide vaccination.

The reasons for the decrease in levels of CSP-specific antibodies following recrudescence/ relapse are unknown. However, it has been extensively reported that infection is associated with immunosuppression,48–50 and it has been demonstrated in rodents51–53 that malaria infection can cause apoptosis of specific T cells, and in humans of mononuclear cells.54

This study of CSP-specific immune responses in individuals recently infected with P. vivax points to an early engagement of the immune system following infection. A long-term follow-up of individuals post-infection may give important insights into the kinetics of CSP-specific immune responses relevant to protection, which will assist our progress in vaccine development.

In summary, the results demonstrate the presence of a significant number of murine T cell epitopes on the P. vivax CSP and a paucity of human T cell responses, possibly as a result of recent infection. In other systems, including the P. falciparum CSP5,9,24,25,27 and the influenza nucleoprotein,29,54,55 there has been a close overlap of murine and human T cell epitopes, both for CD4+ and CD8+ T cell responses. This study may then point to the location of human CD8+ T cell epitopes. It is of concern, however, but not totally unexpected, that of the limited amount of described polymorphisms, variation within P. vivax CSP,16,17 a CTL epitope (v19) was shown to be a site of major polymorphisms (P/T, E/V and K/I; 11; see also Table 1) and that one of the v19 polymorphisms tested was able to abrogate CTL recognition. A detailed knowledge of the specificity and development of CSP-specific immune responses and the likely location of epitopes not readily defined by studying humans naturally exposed to malaria may help in the ultimate design of vaccines incorporating part or all of the CSP.

Table 1

Overlapping peptides representing entire sequence of the circum-sporozoite protein of Plasmodium vivax (Belem and NK and repeat variants of VK)*

Peptide no.ResiduesPeptide sequence
Variant residues are specified in bold.
V11–20MKNFILLAVSSILLVDLFPT
V211–30SILLVDLFPTHCGHNVDLSK
V321–40HCGHNVDLSKAINLNGVNFN
V431–50AINLNGVNFNNVDASSLGAA
V541–60NVDASSLGAAHVGQSASRGR
V646–65SLGAAHVGZSASRGRGLDEN
V756–75ASRGRGLDENPDDEEGDAKK
V866–85PDDEEGDAKKKKDGKKAEPK
V976–95KKDGKKAEPKNPRENKLKQP
V1086–105NPRENKLKQPGDRADGQPAG
V1197–116DRADGQPAGDRADGQPAGDR
V12124–143DRAAGQPAGDRADGQPAGDR
V13160–179DRADGQPAGDRAAGQPAGDR
V14232–251DRAAGQPAGDRAAGQPAGDRC
V15259–278DRAAGQPAGNGAGGQAAGGN
V16NK–insGAGGQAAGGNAANKKAEDAG
V17NK–insAANKKAEDAGGNAGGNAGGG
V18279–298AGGGQGQNNEGANAPNEKSV
V19289–308GANAPNEKSVKEYLDKVRAT
V20299–318KEYLDKVRATVGTEWTPCSV
V21309–328VGTEWTPCSVTCGVGVRVRR
V22319–338TCGVGVRVRRRVNAANKKPE
V23329–348RVNAANKKPEDLTLNDLETD
V24339–358DLTLNDLETDVCTMDKCAGI
V25349–368VCTMDKCAGIFNVVSNSLGL
V26359–378FNVVSNSLGLVILLVLALFN
V27VK247ANGAGNQPGANGAGNQPG-18mer
V28VK247ANGAGNQPGEDGAGNQPG-18mer
V29VK247EDGAGNQPGANGAGNQPG-18mer
V30VK247ANGAGNQPGANGAGGQAA-18mer
Figure 1.
Figure 1.

Proliferative response of undepleted, CD4+ T cell-depleted, and CD8+ T cell-depleted cells in BALB/c (a), B10.D2 (b), B10.BR (c), and B10 (d) mice immunized with Plasmodium vivax peptide pools (see Materials and Methods for details). Data are expressed as delta counts per minute (ΔCPM). The background cpm for the results shown here ranged from 91 to 868.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Figure 2.
Figure 2.

Number of responders by lymphoproliferative response to Plasmodium vivax circumsporozoite protein peptides and antigens in non-exposed individuals (n = 25), in the first malaria episode group (n = 19), and in the multiple episodes group (n = 24). See Materials and Methods for definition of responder. TT = tetanus toxoid; PPD = purified protein derivative of Mycobacterium tuberculosis.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Figure 3.
Figure 3.

a, Cytotoxic T lymphocyte (CTL) activity in BALB/c mice against pooled Plasmodium vivax peptides and P. falciparum peptide (f97). b, CTL activity in B10.D2 mice against pooled P. vivax peptides and P. falciparum peptide (f97). All assays was performed at a 50:1 effector:target ratio.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Figure 4.
Figure 4.

Proliferative responses to P30 (a) and cytotoxic T lymphocyte responses (b). In the lymphoproliferative studies, CD4+ and CD8+ T cells were depleted post-immunization but prior to assay.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Figure 5.
Figure 5.

Cytotoxic T lymphocyte activity against group I Plasmodium vivax circumsporozoite protein peptides in non-exposed- and P. vivax-exposed individuals at different time points. There were 28 subjects in the non-exposed (NE) group, 53 subjects studied at day seven after admission (D7) group, 58 subjects studied at day 28 after admission (D28) group, and 19 subjects studied at follow-up (F) group.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Figure 6.
Figure 6.

Antibody response to a, Plasmodium vivax classic repeat peptide (v14) and b, P. vivax variant repeat peptide (v30) in non-exposed (NE), single and multiply exposed individuals at a serum dilution of 1:100 at different time points. Data are expressed as net optical density at 405 nm (OD405) (see Materials and Methods). Some symbols represent multiple points (see Results for percent of positive responses for each group). For definitions of other abbreviations, see Figure 5.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 1; 10.4269/ajtmh.2004.71.29

Authors’ addresses: Chaisuree Suphavilai, Research Institute for Health Sciences, PO Box 80CMU, Chiang Mai University, Chiang Mai 50202, Thailand. Sornchai Looareesuwan, The Tropical Medicine Hospital, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. Michael F. Good, The Cooperative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, 300 Herston Road, Herston QLD 4006, Queens-land, Australia, Telephone: 61-7-3362-0203, Fax: 61-7-3362-0110, Email: michaelG@qimr.edu.au.

Acknowledgments: We thank the Department of Immunology, Armed Force Research Institute of Medical Sciences (Bangkok, Thailand) for providing tissue culture facilities for the work done in Bangkok. We also thank Dr. Henry Stephens for very helpful information and discussions on HLA class I in Thai population.

REFERENCES

  • 1

    Good MF, Doolan DL, 1999. Immune effector mechanisms in malaria. Curr Opin Immunol 11 :412–419.

  • 2

    Tsuji M, Romero P, Nussenzweig RS, Zavala F, 1990. CD4+ cytolytic T cell clone confers protection against murine malaria. J Exp Med 172 :1353–1357.

    • Search Google Scholar
    • Export Citation
  • 3

    Weiss WR, Sedegah M, Beaudoin RL, Miller LH, Good MF, 1988. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc Natl Acad Sci USA 85 :573–576.

    • Search Google Scholar
    • Export Citation
  • 4

    Hoffman SL, Oster CN, Mason C, Beier JC, Sherwood JA, Ballou WP, Mugambi M, Chulay JD, 1989. Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria. J Immunol 142 :1299–1303.

    • Search Google Scholar
    • Export Citation
  • 5

    Doolan DL, Houghten RA, Good MF, 1991. Location of human cytotoxic T cell epitopes within a polymorphic domain of the Plasmodium falciparum circumsporozoite protein.Int Immunol 3 :511–516.

    • Search Google Scholar
    • Export Citation
  • 6

    Malik A, Egan JE, Houghten RA, Sadoff JC, Hoffman SL, 1991. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. Proc Natl Acad Sci USA 88 :3300–3304.

    • Search Google Scholar
    • Export Citation
  • 7

    Dontfraid F, Cochran MA, Pombo D, Knell JD, Quakyi IA, Kumar S, Houghten RA, Berzofsky JA, Miller LH, Good MF, 1988. Human and murine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein: limited immunogenicity of sporozoites and circumsporozoite protein. Mol Biol Med 5 :185–196.

    • Search Google Scholar
    • Export Citation
  • 8

    Good MF, Kumar S, Weiss WR, Miller LH, 1989. T-cell antigenic sites of the malaria circumsporozoite protein. Blood 74 :895–900.

  • 9

    Kumar S, Miller LH, Quakyi IA, Keister DB, Houghten RA, Maloy WL, Moss B, Berzofsky JA, Good MF, 1988. Cytotoxic T cells specific for the circumsporozoite protein of Plasmodium falciparum.Nature 334 :258–260.

    • Search Google Scholar
    • Export Citation
  • 10

    Avrameas S, Ternynck T, 1969. The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunosorbents. Immunochemistry 6 :53–66.

    • Search Google Scholar
    • Export Citation
  • 11

    Rosenberg R, Wirtz RA, Lanar DE, Sattabongkot J, Hall T, Waters AP, Prasittisuk C, 1989. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax.Science 245 :973–976.

    • Search Google Scholar
    • Export Citation
  • 12

    Franke ED, Lucas CM, Chauca G, Wirtz RA, Hinostroza S, 1992. Antibody response to the circumsporozoite protein of Plasmodium vivax in naturally infected humans. Am J Trop Med Hyg 46 :320–326.

    • Search Google Scholar
    • Export Citation
  • 13

    Park CG, Chwae YJ, Kim JI, Lee JH, Hur GM, Jeon BH, Koh JS, Han JH, Lee SJ, Park JW, Kaslow DC, Strickman D, Roh CS, 2000. Serologic responses of Korean soldiers serving in malaria-endemic areas during a recent outbreak of Plasmodium vivax.Am J Trop Med Hyg 62 :720–725.

    • Search Google Scholar
    • Export Citation
  • 14

    Franke ED, Lucas CM, Roman San E, 1991. Antibody response in humans to the circumsporozoite protein of Plasmodium vivax.Infect Immun 59 :2836–2838.

    • Search Google Scholar
    • Export Citation
  • 15

    Zavala F, Cochrane AH, Naedin EH, Nussenzweig RS, Nussen-zweig V, 1983. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J Exp Med 157 :1947–1957.

    • Search Google Scholar
    • Export Citation
  • 16

    Qari SH, Goldman IF, Povoa MM, Oliveira S, Alpers MP, Lal AA, 1991. Wide distribution of the variant form of the human malaria parasite Plasmodium vivax.J Biol Chem 266 :16297–16300.

    • Search Google Scholar
    • Export Citation
  • 17

    Qari SH, Goldman IF, Povoa MM, di Santi S, Alpers MP, Lal AA, 1992. Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.Mol Biochem Parasitol 55 :105–113.

    • Search Google Scholar
    • Export Citation
  • 18

    Charoenvit Y, Collins WE, Jones TR, Millet P, Yuan L, Campbell GH, Beaudoin RL, Broderson JR, Hoffman SL, 1991. Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. Science 251 :668–671.

    • Search Google Scholar
    • Export Citation
  • 19

    Collins WE, Sullivan JS, Morris CL, Galland GG, Jue DL, Fang S, Wohlhueter R, Reed RC, Yang C, Hunter RL, Lal AA, 1997. Protective immunity induced in squirrel monkeys with a multiple antigen construct against the circumsporozoite protein of Plasmodium vivax.Am J Trop Med Hyg 56 :200–210.

    • Search Google Scholar
    • Export Citation
  • 20

    Thomas BE, Sridevi K, Chopra N, Haq W, Rao DN, 2001. Inducing a cell-mediated immune response against peptides of the Plasmodium vivax circumsporozoite protein. Ann Trop Med Parasitol 95 :573–586.

    • Search Google Scholar
    • Export Citation
  • 21

    Nardin E, Clavijo P, Mons B, van Belkum A, Ponnudurai T, Nussenzweig RS, 1991. T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee. J Immunol 146 :1674–1678.

    • Search Google Scholar
    • Export Citation
  • 22

    Rodrigues MM, Paiva AC, Dutra AP, Yoshida N, Nakaie C, 1991. Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes. Exp Parasitol 72 :271–277.

    • Search Google Scholar
    • Export Citation
  • 23

    Bilsborough J, Carlisle M, Good MF, 1993. Identification of Caucasian CD4 T cell epitopes on the circumsporozoite protein of Plasmodium vivax.J Immunol 151 :890–899.

    • Search Google Scholar
    • Export Citation
  • 24

    Herrera S, Escobar P, de Plata C, Avila GI, Corradin G, Herrera MA, 1992. Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein. J Immunol 148 :3986–3990.

    • Search Google Scholar
    • Export Citation
  • 25

    Zevering Y, Houghten RA, Frazer IH, Good MF, 1990. Major population differences in T cell response to a malaria sporo-zoite vaccine candidate. Int Immunol 2 :945–955.

    • Search Google Scholar
    • Export Citation
  • 26

    Doolan DL, Khamboonruang C, Beck H-P, Houghten RA, Good MF, 1993. Cytotoxic T lymphocyte (CTL) low-responsiveness to the Plasmodium falciparum circumsporozoite protein in naturally-exposed endemic populations: analysis of human CTL response to most known variants. Int Immunol 5 :37–46.

    • Search Google Scholar
    • Export Citation
  • 27

    Blum-Tivouvanziam U, BeghdadiRais C, Roggero MA, Valmori D, Bertholet S, Bron C, Fasel N, Corradin G, 1994. Elicitation of specific cytotoxic T cells by immunization with malaria soluble synthetic polypeptides. J Immunol 153 :4134–4141.

    • Search Google Scholar
    • Export Citation
  • 28

    Houghten RA, 1985. General method for the rapid solid-phase synthesis of large numbers of peptides: Specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA 82 :5131–5135.

    • Search Google Scholar
    • Export Citation
  • 29

    Gotch F, Rothbard J, Howland K, Townsend A, McMichael A, 1987. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326 :881–882.

    • Search Google Scholar
    • Export Citation
  • 30

    Vejbaesya S, Chantangpol R, Longta P, Chandanayingyong D, 1997. HLA class I typing by one-dimensional isoelectric focusing and identification of the new variants in Thai population. Asian Pac J Allergy Immunol 15 :21–27.

    • Search Google Scholar
    • Export Citation
  • 31

    Chiewsilp P, Mongkolsuk T, Sujirachato K, 1997. A*02 in southern Thai Muslims and central Thais. J Med Assoc Thai 80 :S25–S29.

  • 32

    Valmori D, Romero JF, Men Y, Maryanski JL, Romero P, Corradin G, 1994. Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund’s adjuvant (IFA): further enhancement by pre-injection of IFA alone. Eur J Immunol 24 :1458–1462.

    • Search Google Scholar
    • Export Citation
  • 33

    Zevering Y, Khamboonruang C, Rungruengthanakit K, Tungvi-boonchai L, Ruengpipattanapan J, Bathurst I, Barr P, Good MF, 1994. Life-span of human T-cell responses to determinants from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax.Proc Natl Acad Sci USA 91 :6118–6122.

    • Search Google Scholar
    • Export Citation
  • 34

    Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A, 1989. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 19 :2237–2242.

    • Search Google Scholar
    • Export Citation
  • 35

    Weiss WR, Good MF, Hollingdale MR, Miller LH, Berzofsky JA, 1989. Genetic control of immunity to Plasmodium yoelii sporozoites. J Immunol 143 :4263–4266.

    • Search Google Scholar
    • Export Citation
  • 36

    Huang T, Cheng Q, Allan S, Huang Y, 1994. DNA sequencing of circumsporozoite protein genes of Plasmodium vivax from four different countries in west Pacific region: comparative study on the flank sequences. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 12 :85–92.

    • Search Google Scholar
    • Export Citation
  • 37

    Lalvani A, Hurt N, Aidoo M, Kibatala P, Tanner M, Hill AV, 1996. Cytotoxic T lymphocytes to Plasmodium falciparum epitopes in an area of intense and perennial transmission in Tanzania. Eur J Immunol 26 :773.

    • Search Google Scholar
    • Export Citation
  • 38

    Aley SB, Bates MD, Tam JP, Hollingdale MR, 1986. Synthetic peptides from the circumsporozoite protein of Plasmodium falciparum and Plasmodium knowlesi recognize the human hepatoma cell line HepG2-A16 in vitro.J Exp Med 164 :1915–1922.

    • Search Google Scholar
    • Export Citation
  • 39

    Pancake SJ, Holt GD, Mellouk S, Hoffman SL, 1992. Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates. J Cell Biol 117 :1351–1357.

    • Search Google Scholar
    • Export Citation
  • 40

    Rich KA, George FW IV, Law JL, Martin WJ, 1990. Cell-adhesive motif in region II of malarial circumsporozoite protein. Science 249 :1574–1577.

    • Search Google Scholar
    • Export Citation
  • 41

    Rammensee HG, Friede T, Stevanovic S, 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41 :178–228.

  • 42

    Quakyi IA, Currier J, Fell A, Taylor DW, Roberts T, Houghten RA, England RD, Berzofsky JA, Miller LH, Good MF, 1994. Analysis of human T cell clones specific for conserved peptide sequences within malaria proteins. Paucity of clones responsive to intact parasites. J Immunol 153 :2082–2092.

    • Search Google Scholar
    • Export Citation
  • 43

    Zevering Y, Khamboonruang C, Good MF, 1994. Effect of polymorphism of sporozoite antigens on T-cell activation. Res Im-munol 145 :469–476.

    • Search Google Scholar
    • Export Citation
  • 44

    Arevalo-Herrera M, Valencia AZ, Vergara J, Bonelo A, Fleis-chhauer K, Gonzalez JM, Restrepo JC, Lopez JA, Valmori D, Corradin G, Herrera S, 2002. Identification of HLA-A2 restricted CD8(+) T-lymphocyte responses to Plasmodium vivax circumsporozoite protein in individuals naturally exposed to malaria. Parasite Immunol 24 :161–169.

    • Search Google Scholar
    • Export Citation
  • 45

    Zevering Y, Amante F, Smillie A, Currier J, Smith G, Houghten RA, Good MF, 1992. High frequency of malaria-specific T cells in non-exposed humans. Eur J Immunol 22 :689–696.

    • Search Google Scholar
    • Export Citation
  • 46

    Rodrigues M, Nussenzweig RS, Romero P, Zavala F, 1992. The in vivo cytotoxic activity of CD8+ T cell clones correlates with their levels of expression of adhesion molecules. J Exp Med 175 :895–905.

    • Search Google Scholar
    • Export Citation
  • 47

    Braga EM, Carvalho LH, Fontes CJ, Krettli AU, 2002. Low cellular response in vitro among subjects with long-term exposure to malaria transmission in Brazilian endemic areas. Am J Trop Med Hyg 66 :299–303.

    • Search Google Scholar
    • Export Citation
  • 48

    Goonewardene R, Carter R, Gamage CP, Del Giudice G, David PH, Howie S, Kendis KN, 1990. Human T cell proliferative responses to Plasmodium vivax antigens: evidence of immunosuppression following prolonged exposure to endemic malaria. Eur J Immunol 20 :1387–1391.

    • Search Google Scholar
    • Export Citation
  • 49

    Ho M, Webster HK, Looareesuwan S, Supanaranond W, Phillips RE, Chanthavanich P, Warrell DA, 1986. Antigen-specific immunosuppression in human malaria due to Plasmodium falciparum.J Infect Dis 153 :763–771.

    • Search Google Scholar
    • Export Citation
  • 50

    Troye-Blomberg M, Romero P, Patarroyo ME, Bjorkman A, Perlmann P, 1984. Regulation of the immune response in Plasmodium falciparum malaria. III. Proliferative response to antigen in vitro and subset composition of T cells from patients with acute infection or from immune donors. Clin Exp Immunol 58 :380–387.

    • Search Google Scholar
    • Export Citation
  • 51

    Hirunpetcharat C, Good MF, 1998. Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice following challenge with homologous parasite. Proc Natl Acad Sci USA 95 :1715–1720.

    • Search Google Scholar
    • Export Citation
  • 52

    Wipasa J, Xu H, Stowers A, Good MF, 2001. Apoptotic deletion of helper T cells specific for the 19 kDa carboxyl terminal fragment of merozoite surface protein 1 (MSP119) during malaria infection.J Immunol 167 :3903–3909.

    • Search Google Scholar
    • Export Citation
  • 53

    Xu H, Wipasa J, Yan H, Zeng M, Makobongo MO, Finkelman FD, Kelso A, Good MF, 2002. The mechanism and significance of deletion of parasite-specific CD4+ T cells in malaria infection. J Exp Med 195 :881–892.

    • Search Google Scholar
    • Export Citation
  • 54

    Toure-Balde A, Sarthou JL, Aribot G, Michel P, Trape JR, Ro-gier C, Roussilhon C, 1996. Plasmodium falciparum induces apoptosis in human mononuclear cells. Infect Immun 64 :744–750.

    • Search Google Scholar
    • Export Citation
  • 55

    Gould K, Cossins J, Bastin J, Brownlee GG, Townsend A, 1989. A 15 amino acid fragment of influenza nucleoprotein synthesized in the cytoplasm is presented to class I-restricted cytotoxic T lymphocytes. J Exp Med 170 :1051–1056.

    • Search Google Scholar
    • Export Citation
Save