Fine specificities of monoclonal antibodies against the Plasmodium falciparum circumsporozoite protein: recognition of both repetitive and non-repetitive regions

The fine specificities of 6 monoclonal antibodies (MoAbs) raised against the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, were defined by their binding to a series of overlapping octapeptides corresponding to the 7G8 variant of the CS protein. The precise specificities of the MoAbs to the immunodominant NANP repeat region were elucidated by their binding to all possible 4, 5, 6, 7 and 8 amino acid peptides in this region. All 6 MoAbs recognized the NANP repeats. In addition all MoAb bound to nonrepetitive sites with 4 of the 6 MoAbs recognizing known functional sites outside the repeat region including sites required for T cell recognition and hepatocyte invasion. Antibody pressure may therefore be responsible for generating the epitope variation observed at T cell sites. The multiple specificities for all the MoAbs suggests that the repeat region may act as an internal immunological 'smokescreen' by competing more effectively for antibody binding compared to single epitope copy functional sites located outside the repeat region.     

Anti-idiotypic antibodies: preparation and identification; application to the detection of antigens of Plasmodium falciparum

Anti-idiotypic antibodies (anti 94D1Id and anti 94C3Id) were prepared by immunizing rabbits with designated monoclonal antibodies (MoAb) 94D1MoAb (94D1Id) and 94C3MoAb (94C3Id) specific for Plasmodium falciparum. The specificity of both was directed against idiotypes of the relevant MoAb. The interaction between 94D1Id and anti94D1Id or 94C3Id and anti94C3Id was almost completely inhibited when sufficient concentration of P. falciparum antigen was added, suggesting that the idiotypes recognized by anti94D1Id or anti94C3Id were located in the antigen binding site of the MoAbs or the construction of the V region of anti-idiotypic antibodies may be similar to that of P. falciparum antigenic determinants combining with MoAb. Each 4i assay (inhibition of idiotype-anti-idiotype interaction) had a different specificity and sensitivity in detecting P. falciparum asexual antigens. The 94C3Id-anti94C3Id system detected a minimal level of 0.001% parasitaemia and did not cross-react with other species, suggesting that 94C3Id-anti94C3Id system is species specific and may become a valuable tool for immunodiagnosis.     

A high molecular weight antigen in Plasmodium falciparum recognized by inhibitory monoclonal antibodies

Inhibitory monoclonal antibodies which bind to some isolates of Plasmodium falciparum from Papua New Guinea, but not from other areas, bound to a 220 kD antigen. By immunofluorescence microscopy this antigen was shown to be located both within the schizont cytoplasm and also within the schizont infected erythrocyte, but external to the schizont itself. Even at antibody concentrations which caused greater than 70% inhibition of parasite multiplication, accumulation of schizont stages or aggregates of merozoites were not seen, consistent with inhibition occurring at a point after the release of merozoites. While this suggests that the antigen may be present on merozoites, the quantity was below the limit of detection. It is suggested that the large amount of antigen released by rupturing schizonts may be a mechanism used by the parasite to evade immunological attack.     

Topography of epitopes on a polymorphic schizont antigen of Plasmodium falciparum determined by the binding of monoclonal antibodies in a two-site radioimmunoassay

The topographic distribution of common and variant epitopes on two divergent allelic forms of the 185-205K schizont glycoprotein of Plasmodium falciparum were studied by a two-site radioimmunoassay using monoclonal antibodies. Similarities in the conformation of the two molecules were apparent. On both antigens two distinct regions were mapped, each comprising of both strain-common and polymorphic epitopes. Epitopes common to the two PSAs were found to be closely associated with different variable epitopes in tertiary structure. It is suggested that this may contribute to parasite evasion of the host immune response.     

Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen

A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.     

Plasmodium falciparum-inhibitory monoclonal antibodies produced by human hybridomas

Stable human hybridomas were generated that produced inhibitory anti-Plasmodium falciparum monoclonal antibodies. Peripheral blood lymphocytes, obtained from adults in Liberia, a malaria endemic area, were immortalized with Epstein-Barr virus and then fused with KR4, a human, lymphoblastoid cell line. Stable hybridomas that produced anti-P. falciparum monoclonal antibody were identified by an ELISA assay that used the trophozoite and schizont antigens of both the Honduras I and FCR3 parasite strains. Monoclonal antibodies produced by selected hybridomas derived from lymphocytes of two individuals were subsequently studied. The anti-parasite antibodies were produced at 1-3 micrograms/ml in culture supernatants. All of the monoclonal antibodies bound specifically to trophozoites and schizonts of both strains of parasite in an indirect immunofluorescence assay and inhibited production of ring stage parasites by more than 90% when added to trophozoite or schizont containing erythrocytes in culture. Western immunoblot analysis of antigens obtained from trophozoites and schizonts (parasite age span of 36 to 48 h) was performed using either affinity purified or ammonium sulfate-concentrated monoclonal antibody. Antibody from three hybridomas which bound primarily to antigens of the Honduras 1 strain had Mr of approximately 140,000, 130,000 and 123,000.     

Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies, on gametes of different isolates of Plasmodium falciparum

We have studied the properties of epitopes on Plasmodium falciparum gamete surface protein Pfs 48/45, a target antigen of malaria transmission blocking antibodies. Using a two site immunoradiometric assay we have defined three spacially separate, non-repeated, epitope regions on the peptides representing this antigen. Epitope region I is a target of monoclonal antibodies (MoAbs) which strongly suppress infectivity of gametocytes of P. falciparum to mosquitoes; the effect is complement independent and is mediated as effectively by the monovalent Fab fragments as by intact MoAb. Epitope region II consists of two spacially close subregions, IIa and IIb; variant forms of epitopes IIa and IIb occurred in different isolates of P. falciparum. Epitope region III also showed slight structural modification between isolates. MoAbs against regions II or III were relatively ineffective in suppressing gametocyte infectivity compared to MoAbs against region I. However, certain combinations of MoAbs against regions II and III together acted synergistically to suppress infectivity to mosquitoes. All these epitopes failed to react with MoAb when the antigen was presented in reduced form. A fourth epitope, however, was identified which reacted strongly with MoAb when the antigen was presented in reduced form. The MoAb against this epitope had no effect on the infectivity of gametocytes of P. falciparum to mosquitoes.     

Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogeneous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0.01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0.41 with 95% confidence interval 0.20-0.81, P = 0.011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine.     

Allele‐specific antibodies to Plasmodium falciparum merozoite surface protein‐2 and protection against clinical malaria

IgG and IgG3 antibodies to merozoite surface protein‐2 (MSP‐2) of Plasmodium falciparum have been associated with protection from clinical malaria in independent studies. We determined whether this protection was allele‐specific by testing whether children who developed clinical malaria lacked IgG/IgG3 antibodies specific to the dominant msp2 parasite genotypes detected during clinical episodes. We analysed pre‐existing IgG and IgG1/IgG3 antibodies to antigens representing the major dimorphic types of MSP‐2 by ELISA. We used quantitative real‐time PCR to determine the dominant msp2 alleles in parasites detected in clinical episodes. Over half (55%, 80/146) of infections contained both allelic types. Single or dominant IC1‐ and FC27‐like alleles were detected in 46% and 42% of infections respectively, and both types were equally dominant in 12%. High levels of IgG/IgG3 antibodies to the FC27‐like antigen were not significantly associated with a lower likelihood of clinical episodes caused by parasites bearing FC27‐like compared to IC1‐like alleles, and vice versa for IgG/IgG3 antibodies to the IC1‐like antigen. These findings were supported by competition ELISAs which demonstrated the presence of IgG antibodies to allele‐specific epitopes within both antigens. Thus, even for this well‐studied antigen, the importance of an allele‐specific component of naturally acquired protective immunity to malaria remains to be confirmed.     

Protective antibodies against erythrocytic stages of Plasmodium falciparum in experimental infection of the squirrel monkey, Saimiri sciureus

Serum and ascitic fluid from squirrel monkeys ( Saimiri sciureus ) inoculated with erythrocytic stages of Plasmodium falciparum were collected at different periods of the infection. Protection against P. falciparum was achieved by passive transfer of the sera or fluid recovered from animals after spontaneous or drug-induced cure. Purified immunoglobulins from the ascitic fluid also conferred protection. In contrast, protective antibodies directed against erythrocytic stages of P. falciparum could never be demonstrated during the acute phase of infection in spite of the high titres of malarial antibodies detected by immunofluorescence. The comparative immunochemical analysis of antigens recognized by protective and non-protective antibodies revealed quantitative differences which may be of use for the identification of antigens inducing protection.     

恶性疟候选疫苗研究进展

自首次报道减毒子孢子能使人体获得疟疾完全保护力以来,距今已近40年,而有效的疟疾疫苗仍未研制成功。尽管目前疟疾疫苗的研制面临着较大的困难,但恶性疟原虫及冈比亚按蚊全基因序列测定的完成及有效动物模型的建立,给疟疾疫苗的研制带来了希望。该文讨论了过去10年中恶性疟疫苗的研制进展,以期为恶性疟疫苗研发提供新思路。     

Production and characterization of human monoclonal antibodies against Schistosoma mansoni

We have produced a panel of human monoclonal antibodies (MoAb) from patients infected with Schistosoma mansoni in order to analyse more carefully the human immune response to this helminth infection. This study describes the production, characterization and analysis of these MoAbs. Briefly, peripheral blood mononuclear cells from chronically infected patients were (1) isolated and stimulated with parasite antigens in vitro, (2) positively selected for B-cells on anti-Ig columns, and (3) then transformed with Epstein-Barr virus (EBV). Once EBV cell lines were established, they were selected for anti-S. mansoni antibodies using an ELISA, cloned, retested and then fused with the mouse-human heteromyeloma SHM-D33. In this study, we describe five MoAbs which have different antigenic specificities for life-cycle stages based on ELISA to soluble crude antigen preparations, membrane immunofluorescence on whole intact organisms, and immunofluorescent staining of cryostat frozen sections. The importance of these reagents with regard to the human immune response to S. mansoni is currently being evaluated.     

Plasmodium falciparum: use of a NANP19 antibody-test for the detection of infection in non-immune travellers

Circumsporozoite (CS) antibodies are a reliable serological marker for the infection of Plasmodium falciparum. The purpose of this investigation was to construct and evaluate an enzyme-linked immunosorbent assay test for the detection of CS antibodies. While the sensitivity of the newly developed test reached 78%, the specificity was 99%. In addition, the optimized kit was used to test for infection with P. falciparum in 1903 travellers that were recruited from a prospective study for malaria chemoprophylaxis. Sixty-six of the 1903 patients (3.5%) showed elevated CS antigen antibody titres. However, seroconversion could only be demonstrated in 18 (0.95%) patients. Among those seroconverting, there was a significantly higher percentage of male travellers (1.28%) than female travellers (0.56%). Positive reactions were more frequent among returnees from West and East Africa (1.49 and 1.14%, respectively) than among those from other endemic areas, e.g. South America (n=0). Despite its limited sensitivity, this newly developed kit for CS antibody testing may be a valuable tool for the estimation of the risk for travellers in malarious regions to acquire an infection with P. falciparum. It may also be useful for the determination of the efficacy of malaria chemoprophylaxis for inhibiting outbreak of disease.     

多重PCR快速检测恶性疟和间日疟的研究

目的 建立一种简便快速、能同时检测恶性疟和间日疟的核酸检测方法。方法 针对两种疟原虫18S rRNA基因设计2对(3条引物),优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重PCR。并进行最低检测限确定和临床标本检测,以镜检法为金标准分析灵敏度和特异度等指标。结果 该方法可扩增出431 bp(恶性疟原虫)和341 bp(间日疟原虫)基因片段,最低检测限为10²copies/反应,检测临床标本的结果与镜检法无差别(P＞0.05),敏感度为93.55%,特异度为70.83%,阳性预测值为89.23%,阴性预测值为80.95%。结论 所建立的多重PCR方法可快速检测疟疾感染并鉴别分型,灵敏度高,值得推广。     

多糖与恶性疟原虫的分子致病机制

在恶性疟原虫与人体细胞相互作用的过程中,子孢子通过黏附肝内皮细胞受体侵入肝脏,裂殖子通过黏附红细胞表面受体侵入红细胞,感染红细胞利用其表面膜蛋白与人体重要器官的血管内皮细胞表面分子发生黏附,最终导致血流受阻。这些黏附过程均是虫体蛋白与宿主细胞表面带有负电荷的多糖分子相互作用的结果。本文对恶性疟原虫与人体细胞相互作用的分子机制作一综述。     

Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2

There is longstanding evidence for a role of immunoglobulin (Ig)G in protection against malarial disease and infection. IgG1 and IgG3 have been shown to be particularly efficient at associating with monocytes in potentially protective mechanisms (i.e. antibody-dependent cellular inhibition, opsonization and phagocytosis). Conversely, there is some evidence that IgG2 (and possibly IgG4) antibodies may be antagonistic to this protection. The protective effect of IgG subclass antibody activity present before the beginning of a malaria transmission season (preseason antibody levels) against severe malaria has not been tested in longitudinal studies. We measured IgG class and subclass antibody levels specific to crude Plasmodium falciparum lysates by enzyme linked immunosorbent assay in a case-control study of 76 children on the coast of Kenya. The mean optical density values for both IgG class and subclass antibodies were not significantly different between the children who developed severe malaria and those who remained healthy during an observation period of two malaria transmission seasons. However, elevated levels of IgG1 in relation to levels of IgG2 and IgG4 antibodies were associated with protection from severe malaria (P = 0.02). Conversely, elevated levels of IgG2 in relation to IgG1 and IgG3 antibodies were associated with a higher risk of developing severe malaria (P = 0.006).     

Immunization against Plasmodium falciparum with recombinant polypeptides produced in Escherichia coli

Two proteins produced in recombinant Escherichia coli and containing amino acid sequences from the Plasmodium falciparum precursor to major merozoite surface antigens (PMMSA) have been partially purified. These proteins, together with a preparation of merozoites, have been used to immunize animals. The antibody response and the degree of protection were compared. Animals immunized with merozoites produced antibodies reacting with many P. falciparum proteins, whereas a response specific for PMMSA was detected in those receiving the recombinant material. Incomplete protection was conferred to both groups and there was no apparent correlation between antibody levels and protection.     

Human antibody responses to Pfs 230, a sexual stage-specific surface antigen of Plasmodium falciparum: non-responsiveness is a stable phenotype but does not appear to be genetically regulated

The 230 kD gamete surface protein of the malaria parasite Plasmodium falciparum (Pfs 230) is a target of transmission blocking antibodies. Anti-Pfs 230 antibodies are induced following natural infection with malaria but are not found in all P. falciparum-exposed individuals. In this study we have shown that approximately 40% of malaria-exposed Gambians do not make antibodies to the native Pfs 230 molecule. This phenotype is remarkably stable over time and does not appear to be related to age, malaria exposure or major histocompatibility complex genotype. Comparison of antibody responses in twins indicates that the anti-Pfs 230 response is not strictly genetically controlled, but a high degree of concordance within both dizygous and monozygous twin pairs suggests that factors associated with exposure to malaria in childhood may be important in determining the subsequent immune response.     

Monkey-derived monoclonal antibodies against Plasmodium falciparum.

A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen.