Monoclonal antibodies to stage-specific, species-specific, and cross-reactive antigens of the rodent malarial parasite, Plasmodium yoelii.

Eighteen hybridoma cell lines were used to study species-specific, stage-specific, and serological cross-reactive antigens of the rodent malarial parasite, Plasmodium yoelii. Specificity and location of plasmodial antigens were determined by indirect fluorescent-antibody analysis. Results showed that a minimum of 12 distinct plasmodial antigens could be distinguished by the 18 hybridomas. Antigens were found on the surface or within the cytoplasm of the parasite, but not on the surface of erythrocytes from infected animals. The majority (11 of 12) of antigens were present in all erythrocytic stages of the parasite, but one was stage-specific for merozoites. Additional studies showed that 6 of 18 of the monoclonal antibodies identified species-specific antigens, 2 of 18 recognized antigens confined to related rodent malarial parasites (Plasmodium berghei, Plasmodium vinckei, and Plasmodium chabaudi), whereas 8 of 18 detected cross-reactive antigens common to rodent, primate (Plasmodium knowlesi, Plasmodium falciparum), and avian (Plasmodium gallinaceum) malarias.     

Cytokine-induced inhibition of Plasmodium falciparum erythrocytic growth in vitro.

The addition of recombinant cytokines to Plasmodium falciparum in vitro cultures retarded the growth of the parasite with the effect of recombinant IL-2 (rIL-2) > interferon-gamma (IFN-gamma) > tumour necrosis factor-beta (TNF-beta). The process was concentration dependent, being greatest at 30,000 U/ml and required a 72-h period of continuous exposure for maximum effect. Growth inhibition, as determined morphologically and radiometrically, was a consequence of defective schizont maturation rather than inhibition of merozoite invasion. It was cumulative and detectable within one erythrocytic (48 h) growth cycle.     

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms

The immunogenicity of the carrier-free synthetic peptide, (NANP)₄₀, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2^b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)₄₀ IgG antibodies. This decrease in the anti-(NANP)₄₀ antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)₄₀ peptide, and on an already established anti-(NANP)₄₀ antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)_n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.     

Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro.

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.     

Brefeldin A inhibits protein secretion and parasite maturation in the ring stage of Plasmodium falciparum.

The release of all newly synthesized soluble proteins from the ring stage of Plasmodium falciparum-infected erythrocytes was reversibly blocked by brefeldin A, indicating the presence of a conserved step of classical eukaryotic secretory export within the parasite. This implies that proteins exported to the erythrocyte cytosol undergo secretory release at the parasite plasma membrane and subsequent translocation across the vacuolar membrane. Along with inhibiting protein export brefeldin arrested parasite maturation, but the cells remained viable even after 24 h in the presence of the drug. The results suggest that secretory export may be important for development, but not for immediate survival, at the ring stage.     

Plasmodium chabaudi chabaudi and P. falciparum : inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin

The major leucine aminopeptidase of the rodent malarial parasite Plasmodium chabaudi chabaudi was partially purified using a combination of high-pressure liquid chromatography on a size-exclusion column and affinity chromatography using the aminopeptidase-specific inhibitor bestatin as the ligand. The purified enzyme showed simple Michaelis-Menten kinetics when the fluorogenic peptide analogue leucyl-7-amino-4-methyl-courmarin served as the substrate, and it was strongly inhibited by both bestatin (K _i= 50.7 ± 21.0 nM) and nitrobestatin (K _i= 2.51 ± 0.2 nM) in a competitive manner. These inhibitors were also potent blockers of the growth of P. c. chabaudi and the human parasite P. falciparum in culture, and nitrobestatin was again the more potent. Therefore, the leucine aminopeptidase represents an important target to which novel anti-malarial agents could be directed. Received: 3 November 1997 / Accepted: 21 January 1998     

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.     

Familial correlation of immunoglobulin G subclass responses to Plasmodium falciparum antigens in Burkina Faso

Host genes are thought to determine the immune response to malaria infection and the outcome. Cytophilic antibodies have been associated with protection, whereas noncytophilic antibodies against the same epitopes may block the protective activity of the protective ones. To assess the contribution of genetic factors to immunoglobulin G (IgG) subclass responses against conserved epitopes and Plasmodium falciparum blood-stage extracts, we analyzed the isotypic distribution of the IgG responses in 366 individuals living in two differently exposed areas in Burkina Faso. We used one-way analysis of variance and pairwise estimators to calculate sib-sib and parent-offspring correlation coefficients, respectively. Familial patterns of inheritance of IgG subclass responses to defined antigens and P. falciparum extracts appear to be similar in the two areas. We observed a sibling correlation for the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against ring-infected-erythrocyte surface antigen, merozoite surface protein 1 (MSP-1), MSP-2, and P. falciparum extract. Moreover, a parent-offspring correlation was found for several IgG subclass responses, including the IgG, IgG1, IgG2, IgG3, and IgG4 responses directed against conserved MSP-2 epitopes. Our results indicated that the IgG subclass responses against P. falciparum blood-stage antigens are partly influenced by host genetic factors. The localization and identification of these genes may have implications for immunoepidemiology and vaccine development.     

Antibody-independent inhibition of Plasmodium falciparum in vitro cultures.

The sera of 100 Colombian individuals of African origin living in a malaria-endemic area of the Pacific coast were studied with regard to their capacity to inhibit Plasmodium falciparum cultures in vitro. Antimalarial antibody levels determined by indirect immunofluorescence were higher in the group of infected individuals than in the noninfected individuals, and inhibitory activity assessed by the inhibition of parasite incorporation of 3H-hypoxanthine in vitro was present in the sera of both the infected and noninfected patients. We believe that the noninfected patients were probably immune. The sera of some of the infected patients had high inhibitory capacities for the P. falciparum FCB-1 isolate. When the inhibitory effects of some of the sera were tested by using four parasite isolates from different regions of the world, striking differences among them were found.     

Regulation of antigen-specific immunoglobulin G subclasses in response to conserved and polymorphic Plasmodium falciparum antigens in an in vitro model

Cytophilic antibodies (Abs) play a critical role in protection against Plasmodium falciparum blood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed to P. falciparum or unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP1(19)], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. No P. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently to P. falciparum infections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP1(19), R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.     

Antimalarial antibodies of the immunoglobulin G2a isotype modulate parasitemias in mice infected with Plasmodium yoelii.

Previous studies have demonstrated the importance of antibodies in mediating immunity to malaria, but the relative contribution of the different immunoglobulin isotypes has not been assessed. In this study, hyperimmune plasma was generated against Plasmodium yoelii and separated by protein A-Sepharose chromatography into fractions containing immunoglobulin G1 (IgG1), IgG2a, IgG2b, or IgG3 antibodies and the remaining nonbinding plasma proteins, including IgM. Following concentration, the antimalarial titer of each isotypic fraction was approximately equivalent to the corresponding isotype in hyperimmune plasma. The isotypic fractions were passively transferred to BALB/c and outbred ICR mice prior to challenge with virulent P. yoelii 17XL and to CBA/CaJ mice challenged with avirulent P. yoelii 17XNL. Only mice receiving IgG2a antibodies experienced an altered course of infection. Immunoprecipitation studies showed that all four IgG isotypes appear to recognize a similar set of antigens. These results suggest that antimalarial antibodies of the IgG2a isotype play a dominant role in modulating P. yoelii parasitemias.     

Placental Plasmodium falciparum infection: causes and consequences of in utero sensitization to parasite antigens

Available evidence suggests that, in African populations, systemic blood-dwelling parasitoses of mothers are associated with enhanced susceptibility to infection of their offspring. Thus, children born to mothers with filariasis or schistosomiasis are infected earlier, and offspring of mothers with placental Plasmodium falciparum at delivery, commonly referred to as pregnancy-associated malaria or PAM, are themselves at higher risk of developing parasitaemia during infancy. Since foetal/neonatal antigen-presenting cells (APC) are either immature or provide insufficient costimulatory signals to T cells, thus favouring tolerance induction, it is commonly assumed that soluble parasite components [protein antigens], transferred transplacentally and inducing foetal immune tolerance, are largely, if not exclusively, responsible for these outcomes. Plasmodial asexual blood stage antigen-specific T cells are detectable in as many as two-thirds of all cord blood samples in malaria-endemic countries of sub-Saharan Africa, indicating that in utero sensitization may be a common phenomenon during pregnancy in these populations. Parasite antigen-specific T cell responses of neonates born to helminth-infected mothers display a highly skewed Th2-type cytokine pattern, with a prominent role for the regulatory cytokine interleukin (IL)-10. Similarly, the cord blood immune response of those born to mothers identified with on-going PAM is characterised by inducible parasite antigen-specific IL-10-producing regulatory T cells that can inhibit both APC HLA expression and Th1-type T cell responses. In contrast, plasmodial antigen-specific Th1-type responses, characterised by IFN-gamma production, predominate in cord blood of those born to mothers successfully treated for Pf malaria during gestation, suggesting that the duration and/or the nature of antigen exposure in utero governs the outcome with respect to neonatal immune responses. Aspects of APC function in the context of these differentially modulated responses, whether and how the latter translate into altered susceptibility to Pf infection during infancy, as well as the possible implications for vaccination in early life, are aspects that are discussed in this review.     

Immunity to blood stages of Plasmodium falciparum is dependent on a specific pattern of immunoglobulin subclass responses to multiple blood stage antigens

BACKGROUND: In view of the lack of effectiveness of vaccines against Plasmodium falciparum malaria new approaches in the search for antigens and formulations for a vaccine are required. Immunoglobulin responses against several blood stage antigens were only partially associated with protection from symptoms in prospective immuno-epidemiological studies. HYPOTHESIS: Immunity to blood stages of P. falciparum is dependent on a specific pattern of immunoglobulin subclass responses to multiple blood stage antigens. SUPPORTING EVIDENCE FOR THE HYPOTHESIS: This hypothesis results from previous studies showing that IgG1 and IgG3 responses against antigens like ring-infected erythrocyte surface antigens, merozoite surface proteins and variant surface antigens on schizonts were associated with clinical immunity against malaria. None of the specific responses against a single antigen was completely protective. IMPLICATIONS OF THE HYPOTHESIS: A confirmation of the hypothesis would support efforts to generate a P. falciparum vaccine containing a comprehensive set of blood stage antigens, which, using adjuvant technology, would lead to the appropriate immunoglobulin subclass response. MEANS TO TEST THE HYPOTHESIS: Detection of multiple immunorelevant P. falciparum blood stage antigens could be achieved by two-dimensional electrophoresis and blotting of the antigens onto nitrocellulose followed by exposure to the participant's serum. Bound immunoglobulins are visualized by isotype specific peroxidase conjugated anti-human immunoglobulin and quantified by transmission densitometry. The resulting pattern of responses for each immunoglobulin isotype could be compared between clinically immune participants remaining asymptomatic with subsequent P. falciparum infections and people who develop malaria. Immunorelevant antigens are identifiable by mass spectrometry with the fully decoded P. falciparum genome.     

Rhoptry-associated protein 1-binding monoclonal antibody raised against a heterologous peptide sequence inhibits Plasmodium falciparum growth in vitro

Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-1(23-711)) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-1(23-711) MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.     

Differential effect of immunoglobulin on the in vitro growth of several isolates of Plasmodium falciparum.

Immunoglobulin isolated from the sera of individuals living in a malarious area of Papua New Guinea was tested for an effect on the growth in vitro of four isolates of Plasmodium falciparum, three from Papua New Guinea and one from Thailand. The Papua New Guinea isolates were inhibited to the same degree by individual immunoglobulin preparations, and inhibition varied from 0 to 98% (assessed by a radioisotopic readout). Immunoglobulin preparations which inhibited the Papua New Guinea isolates caused less inhibition of the Thai parasites. Biosynthetically labeled parasite proteins were analyzed by two-dimensional gel electrophoresis, and differences were detected in the protein and antigenic composition of isolates which differed in their sensitivity to inhibitory immunoglobulin. Three acidic proteins (Mr 200,000, 150,000, and 65,000) were found only in the Papua New Guinea isolates. All Papua New Guinea isolates contained a high-molecular-weight basic protein with an Mr of 220,000 (Pf220), but the corresponding protein of the same molecular weight in the Thai isolate had a more acidic isoelectric point. Another isolate (from Africa) initially showed a degree of resistance to inhibition by Papua New Guinea immunoglobulin (although not to the same extent as the Thai parasite), but in later experiments, this isolate was susceptible to inhibition. During the course of this series of experiments, the antigenic composition of this (uncloned) isolate changed so that it became similar (but not identical) to the Papua New Guinea isolates.     

Use of a monoclonal, anti Plasmodium berghei antibody, cross reacting with P. falciparum, for the detection of P. falciparum in in vitro infected blood

This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.     

Wild isolates of Plasmodium falciparum malaria show decreased sensitivity to in vitro inhibition of parasite growth mediated by autologous host antibodies

Antigenic diversity in field populations of Plasmodium falciparum parasites may delay the acquisition of protective immunity to malaria, the development of which may thus require repeated exposure to infection over a prolonged period of time. In this study we show that P. falciparum parasites may vary in their sensitivity to antibody-mediated invasion/growth inhibition in vitro. Wild isolates of P. falciparum from children living in an endemic area of Burkina Faso were tested for their sensitivity to the growth inhibitory effects of antibodies originating from the same (autologous) and from other donors (heterologous). A significantly lower invasion inhibition activity was obtained when the isolates and antibodies were tested in autologous compared with heterologous combinations. The lower sensitivity to growth inhibition by autologous antibodies may be due to immune pressure in vivo, selecting from a heterogeneous parasite population those with a low expression of the antigens recognized by the host's antibodies. Alternatively, the parasites cultured from each child might represent expanding parasite populations, mainly constituting strains not earlier seen by the immune system of that specific host. The results reinforce the concern about Plasmodium antigenic diversity as a major obstacle towards the development of an effective malaria vaccine.     

T-cell recognition of a cross-reactive antigen(s) in erythrocyte stages of Plasmodium falciparum and Plasmodium yoelii: inhibition of parasitemia by this antigen(s).

In the current study, we investigated the presence of a cross-reactive antigen(s) in the erythrocyte stage from Plasmodium yoelii (265 BY strain) and Plasmodium falciparum through recognition by T cells primed in vivo with antigens from each of these parasites. BALB/c mice are naturally resistant to P. falciparum but are susceptible to P. yoelii infection. Mice that had recovered from P. yoelii primary infection became resistant to a second infection. A higher in vitro proliferative response to a soluble blood stage preparation of P. falciparum was observed in splenic cells from immune animals than in those from mice with a patent P. yoelii infection. The antigen-induced proliferative response was enhanced when animals were exposed to a secondary infection. Animals exposed to a challenge infection were treated with anti-CD4 or anti-CD8 monoclonal antibodies to deplete the corresponding subset of T cells. There was a marked diminution in P. falciparum antigen-induced proliferative response in the total splenic cell populations from CD8-depleted but not from CD4-depleted mice. In CD8-depleted and nondepleted animals, the antigen-induced proliferation in the total cell populations was markedly lower than in the T-cell-rich populations, indicating inhibitory activities of B cells and/or macrophages. There was no such difference in the stimulation between total and T-enriched cell populations from CD4-depleted animals. Flow cytometry analysis demonstrated the presence of an almost equal percentage of CD8+ (59.6%) and CD4+ (64%) T cells in the spleen preparations following in vivo depletion of CD4- and CD8-bearing T cells, respectively. When cultured with P. yoelii blood stage antigen, splenocytes from animals immunized with P. falciparum antigen displayed a significant proliferative response which was markedly diminished by treatment with anti-Thy-1.2 antibody plus complement. Animals immunized with P. falciparum antigen and then challenged with P. yoelii blood stage parasites displayed about a 50% lower level of parasitemia. These results demonstrated the existence of a cross-reactive antigen(s) between a murine and a human Plasmodium species, as determined from both in vivo and in vitro biological assays, and indicated the reactivity of mainly CD8+ T cells with this antigen.     

Effector cells involved in nonspecific and antibody-dependent mechanisms directed against Plasmodium falciparum blood stages in vitro.

We have evaluated in in vitro conditions the possible cooperative effect of antimalarial antibodies with several human blood cell types. When used alone, immunoglobulin G from African adults who had reached a state of premunition against malaria was found to have no or very limited direct effect on invasion and multiplication of P. falciparum asexual blood stages. In contrast, these antibodies induced a marked specific inhibition of parasite growth in the presence of normal blood monocytes, and the inhibition did not appear to be strain dependent. No similar antibody-dependent cellular inhibitory effect was found using human blood polymorphonuclear leukocytes, lymphocytes, platelets, or adherent spleen cells. However, these cells could all exert in vitro some non-antibody-dependent inhibitory effect when present at high effector/target cell ratios.