Monoclonal antibodies produced against sporozoites of the human parasite Plasmodium malariae abolish infectivity of sporozoites of the simian parasite Plasmodium brasilianum.

We have used a sporozoite neutralization assay to define the biological relevance of the cross-reactivity of two monoclonal antibodies, raised against sporozoites of the human parasite Plasmodium malariae (Uganda 1/CDC), with sporozoites of the simian parasite Plasmodium brasilianum (Colombian). In vitro incubation of each of these two monoclonal antibodies with sporozoites of P. brasilianum totally abolished the infectivity of these parasites for Saimiri sciureus. Using Western blot analysis and one of the P. malariae monoclonal antibodies, we identified two sporozoite proteins characteristic of the Colombian isolate of P. brasilianum with apparent molecular weights of 56,000 and 66,000. The same monoclonal antibody identified two proteins in an extract of the Peruvian isolate of P. brasilianum with apparent molecular weights of 59,000 and 69,000.     

A rapid inhibition micro ELISA for detecting antibodies to Plasmodium falciparum sporozoites in human blood

A sensitive and specific micro ELISA, named MONOPLATE ELISA, for the detection of antibodies against P. falciparum sporozoites was developed. It can be applied to many kinds of samples including serum, plasma, whole blood, eluted bloodspot and mosquito bloodmeal as well. The method makes use of a single microtiter plate and the chemically synthesized (Asn-Ala-Asn-Pro)20 (NANP20) antigen both as coating material and as competitive (binding) inhibitor in the samples. The specific value of each sample is obtained as the absorbance difference between the uninhibited and the fully inhibited sample. Using appropriate conditions, the results can be evaluated by simple visual inspection of the plate, without any instrument. A rapid procedure, where the incubation times for sample and conjugate are just 15 minutes, is also described. When unknown samples from a P. falciparum endemic area were tested, a close correlation was found between our results and those obtained with the only commercial ELISA kit now available (Sclavo S.p.A). For screening purposes, as many as 48 samples per plate can be tested by this method.     

Characterization of antibodies to sporozoites in Plasmodium falciparum malaria and correlation with protection.

The antibody response to sporozoites of Plasmodium falciparum and the role of these antibodies in protection against malaria have not been systematically investigated. An understanding of antisporozoite antibodies in natural infection is, however, important to the development of a human malaria vaccine. In a prospective study in Thailand, an antibody response to sporozoites was observed only in individuals who developed parasitemia. Antibodies were detected against an epitope in the repeat region of the circumsporozoite (CS) protein. Current candidate sporozoite vaccines are based on CS repeat antigens. The CS antibody response was of low magnitude, peaked after detection of parasitemia, and had a serum half-life of less than 1 month. CS antibody boosting occurred in only 6% of reinfected individuals. These observations suggest that antisporozoite antibody is poorly developed under natural conditions and appears not to protect against development of malaria.     

Synthetic peptides as antigens for the detection of humoral immunity to Plasmodium falciparum sporozoites

The presence of antibodies against P. falciparum sporozoites in humans living in malaria-endemic areas was measured using as antigen the synthetic peptide (NANP)3, which represents the immunodominant region of the circumsporozoite (CS) protein. By using a competitive binding assay it was determined that antibodies which recognize (NANP)3 do not react with a 22-Mer synthetic peptide representing a cross-reacting epitope present in an antigen (5.1) from the blood stages of the parasite. Antibodies present in human sera which react with the 5.1 peptide did not react with (NANP)3. This strongly suggests that antibodies to (NANP)3 found in sera of individuals living in endemic areas are a reflection of exposure to P. falciparum sporozoites. These results validate the use of (NANP)3 for epidemiological studies to detect and measure humoral immunity to P. falciparum sporozoites.     

Detection of anti-neutrophil cytoplasmic antibodies after acute Plasmodium falciparum malaria.

Four of 30 patients with Plasmodium falciparum infection in Bangkok, Thailand, were positive for anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence 1 month after antimalarial therapy. No myeloperoxidase, proteinase 3, lactoferrin, or elastase reactivity was found. Since no evidence of vasculitis was seen in these patients, anti-neutrophil cytoplasmic antibody production in malaria-infected susceptible patients probably represents a secondary response, indicating neutrophil activation.     

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.     

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein.

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.     

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.     

Inhibitory activity against sporozoites induced by antibodies directed against nonrepetitive regions of the circumsporozoite protein of Plasmodium vivax.

In previous studies, Saimiri sciureus boliviensis monkeys have been immunized with four recombinant proteins reproducing part of the circumsporozoite (CS) protein of Plasmodium vivax sporozoites (NS1(81) V20, rPvCS-1, rPvCS-2, rPv-CS-3), or with irradiated sporozoites of P. vivax Salvador I strain. To analyze the antibody response elicited against epitopes located outside the immunodominant repeat region of the CS protein, serum samples from these animals were tested for their ability to inhibit the in vitro development of liver stages of P. vivax VK247 strain, characterized from the other strains only by a specific repeat region on the CS protein. Results indicated that there is at least one protective B-cell epitope outside the repeat region of the CS protein of P. vivax sporozoites, and that this epitope can be expressed by irradiated sporozoites, rPvCS-1 and -3, but not by rPvCS-1 or NS1(81)V20. Therefore, we designed peptides from the amino acid sequences present both in rPvCS-2 and -3, but not included in the recombinant proteins rPvCS-1 and NS1(81)V20. Anti-peptide antibodies had no activity on the development of sporozoites of P. vivax Salvador I strain, into schizonts in primary culture of Saimiri monkey hepatocytes. In addition, antisporozoite antibodies did not react with any of the peptides. These results suggest that the in vitro inhibition observed in this study could depend upon the conformation of the CS protein. This study also demonstrates that antibody response to unnatural linear epitopes can be induced by immunization with recombinant proteins.     

Immunization against Plasmodium falciparum asexual blood stages using soluble antigens.

Saimiri sciureus monkeys have been successfully immunized against a human malaria parasite, Plasmodium falciparum, using soluble antigens purified from schizont and merozoite extracts. High levels of antibodies reacting with schizont and merozoite specific polypeptides of 140 and 200 kdaltons were detected in the sera of protected monkeys. The five immunized monkeys survived a challenge infection with 5 X 10(7) parasites inducing a fulminant disease in control monkeys.     

Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.     

Detection of E. coli colonies expressing the v-sis oncogene product with monoclonal antibodies made against synthetic peptides

A method for immunodetection of individual epitopes on eukaryotic proteins synthesized in E. coli colonies is described. The system is developed using monoclonal antibodies produced against the Ha-ras protein produced in E. coli JM103. Monoclonal antibodies made against a synthetic peptide from the v-sis oncogene sequence are then used to identify bacterial colonies in which the v-sis protein is being produced. Production of the v-sis protein by these E. coli colonies was confirmed by immunoblot analysis. The assay utilizes peroxidase conjugated anti-mouse immunoglobulin and 4-chloro-1-naphthol to detect the positive colonies and can detect on the order of 200 pg antigen per E. coli colony.     

Studies on the humoral immune response to a synthetic vaccine against Plasmodium falciparum malaria.

A synthetic vaccine against the asexual blood stages of P. falciparum, the SPf 66 synthetic hybrid polymer, composed of peptides derived from three merozoite membrane proteins as well as one peptide from the sporozoite CS protein, has been developed by our group and tested in different protection assays in Aotus monkeys as well as in human volunteers. This study evaluates the humoral immune response induced by the SPf 66 protein vaccination in adult human volunteers from the Colombian Pacific coast as follows: determination of specific IgG antibody levels against SPf 66 by FAST-ELISA after each immunization; analysis of antibody reactivity with P. falciparum schizont lysates by immunoblots; and determination of the in vitro parasite growth inhibition. A clear boosting effect, dependent on time and dose, was observed in the antibody production kinetics. These antibodies also specifically recognize three proteins of the P. falciparum schizont lysate corresponding to the molecular weights of the proteins from which the amino acid sequence was derived. These sera were also capable of markedly inhibiting in vitro parasite growth.     

Detection of canine parvovirus antigens with antibodies to synthetic peptides

Summary Antibodies produced in rabbits against an 18-amino acid peptide (peptide 1, NSLPQSEGATNFGDIGVP) of capsid protein VP2/residues 292–309 of canine parvovirus (CPV) or against an 18-amino acid peptide (peptide 2, GKRNTVLFHGPASTKGKS) of nonstructural protein NS1/residues 391–409 of CPV identified, in immunofluorescence analysis, viral antigens in canine A 72 cells infected with CPV. Antibodies to peptide 2 also identified viral antigens in bovine cells infected with bovine parvovirus. In western blot analysis, antibodies to peptide 1 and peptide 2 also detected viral antigens derived from blue fox parvovirus, feline parvovirus, mink enteritis virus and raccoon dog parvovirus. The peptide antibodies could be used as convenient tools in diagnosis of infections caused by CPV or closely related viruses affecting cats, minks, blue foxes and raccoon dogs.     

T-cell receptors of man and mouse studied with antibodies against synthetic peptides.

We used polyclonal rabbit antibodies directed against synthetic peptides predicted from the gene sequence of the human T-cell receptor (TCR) beta-chain YT35 to study the antigen receptor on human helper T-cell leukemia lines and on normal mouse thymocytes. Antibodies were raised to peptides corresponding to joining segment (J beta) and to a conserved stretch of sequence around the first cysteine in the constant region (C beta). These peptides were selected on the basis of homology with corresponding segments of immunoglobulin light chains. The specificity of the antibodies was established using synthetic overlapping peptides that modelled the complete TCR beta-chain. Western blot analysis was performed against detergent lysates of T cells. Both of the antibodies reacted strongly with 2-3 polypeptides in the mass range 40-45 kDa in mouse and human cells. Clearance experiments using monoclonal antibodies against murine TCR alpha- and beta-chains and against human TCR beta-chain and immunoprecipitations with monoclonal antibody to the murine T3 complex established that these components represented the alpha/beta heterodimer. An additional component around 31 kDa was detected by anti-J beta antibodies in murine thymus extracts. The use of the affinity-purified antipeptide antibody in two-dimensional Western blot analyses allows the clear discrimination between the characteristic individual receptors of monoclonal neoplastic T cells and the polydisperse patterns representative of heterogeneous normal populations. Antigenic cross-reactions between T-cell receptor beta-chains of man and mouse observed with monoclonal antibodies and rabbit antisera to peptides are consistent with the homology in gene sequence between the two species.     

Detection of antigens and antibodies in the urine of humans with Plasmodium falciparum malaria.

Humans infected with Plasmodium falciparum frequently have elevated levels of proteins in their urine, but it is unclear if any of these proteins are parasite antigens or antimalarial antibodies. To resolve this question, urine samples from malaria patients and controls living in Thailand and Ghana were evaluated. Urine samples from 85% of the patients had elevated protein levels and contained proteins with Mrs ranging from less than 29,000 to greater than 224,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antisera were produced against urine from infected and control subjects. Antisera raised against infected, but not control, urine were positive by indirect immunofluorescence on P. falciparum parasites and immunoprecipitated approximately 12 unique bands from extracts of parasites metabolically labeled with 35S-methionine. These data suggest that a variety of P. falciparum antigens are released into urine during acute infection. It is also likely that anti-P. falciparum antibodies are present in the urine of malaria patients because samples from these patients, but not controls, were positive in indirect immunofluorescence assays and immunoprecipitated at least 19 P. falciparum antigens from extracts of metabolically labeled parasites. The detection of malarial antigens and antibodies in urine may lead to a new approach for the diagnosis of malaria.     

Induction of humoral immune response against Plasmodium falciparum sporozoites by immunization with a synthetic peptide mimotope whose sequence was derived from screening a filamentous phage epitope library.

The mouse monoclonal antibody 2A10 (immunoglobulin G), which recognizes the (NANP)n repeat of Plasmodium falciparum circumsporozoite surface protein, was used to screen a filamentous phage epitope library expressing random amino acid hexamers. The sequences obtained were TNRNPQ, SNRNPQ, NND-NPQ, SNYNPQ, and QNDNPQ (single-letter amino acid designation). These peptides showed 50% homology with the native epitope (PNANPN) and therefore were considered to mimic its structure (mimotopes). Two of these mimotopes (TNRNPQ and NNDNPQ) inhibited the binding of monoclonal antibody 2A10 to the recombinant protein R32LR, which contains the amino acid sequence [(NANP)15NVDP]2. Immunization of mice and rabbits using the peptide (TNRNPQ)4 induced a humoral response that recognized R32LR by an enzyme-linked immunosorbent assay and P. falciparum sporozoites by an immunofluorescence assay. These results suggest that phage epitope libraries can be exploited to screen for mimotopes in the design of subunit vaccines against infectious agents.     

Anti-phospholipid antibodies in patients with Plasmodium falciparum malaria.

Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease.