Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.     

Naturally acquired immunity to Plasmodium falciparum

Malaria infections induce multiple humoral and cellular responses, most of which are probably not protective. This discussion of the epidemiology of acquired immunity to malaria will concentrate on two main areas: first, the relationship between parasitism and disease in endemic settings and the constraints placed on determining which responses are important in acquired protective immunity; second, the central importance of antigenic diversity in the host-parasite relationship. The emphasis throughout, unless otherwise stated, will be on the major human pathogen Plasmodium falciparum.     

Histocompatibility antigens and humoral immunity to Epstein-Barr virus

To explore possible genetic determinants of immunity to Epstein-Barr (EB) virus infection, prevalence rates and geometric mean titers (GMT) of antibody against EB viral capsid antigen (VCA) were measured in 422 adults of known HLA and ABO histocompatibility types. Deviation from the overall prevalence (87%) of anti-VCA titers > 10 was limited to subjects with HLA-A10 (95%) and those with a "blank" HLA-A locus (78%). The latter deviation was largely accounted for by the 13 subjects of HLA-A1/blank constitution (46%). Deviations from the overall mean anti-VCA titer (GMT 131.5) were found in seropositive subjects with blood group 0 (GMT 153.3) and tissue antigens HLA-A3 (GMT 150.8), HLA-B7 (GMT 152.1), HA-Bw15 (GMT 97.0), and HLA-B27 (GMT 86.4). Separate reanalysis of the data for male and female subjects verified the association of blood group 0 with elevated and HLA-B27 with reduced mean levels of anti-VCA antibody.     

Inhibitory monoclonal antibodies to soluble Plasmodium falciparum antigens

Several murine monoclonal antibodies were raised against purified soluble Plasmodium falciparum antigens from the asexual blood stage. The monoclonal antibodies were purified from ascites by preparative agarose gel electrophoresis and tested for inhibitory activities against P. falciparum in vitro cultures. One monoclonal antibody, HATR 2–4, showed an isolate-specific growth inhibition of P. falciparum in vitro cultures. The antibody reacted in immunoblotting with bands of 250 and 57 kilo dalton (kdalton). Another monoclonal antibody, HATR 2–8, showed growth inhibition of several geographically distinct P. falciparum isolates. HATR 2–8 reacted in immunoblotting with bands of 250 and 74 kdalton. Heating of the antigens destroyed the reactivity of HATR 2–8. The monoclonal antibodies HATR 2–4 and HATR 2–8 probably recognize different epitopes on the same antigen. This antigen circulates in the plasma of some patients with P. falciparum parasitaemia.     

Induction of protective immunity to monoclonal-antibody-defined Plasmodium falciparum antigens requires strong adjuvant in Aotus monkeys.

Monoclonal antibodies to the major Plasmodium falciparum merozoite surface coat and rhoptry antigens were produced. A combination of the affinity-purified polypeptides with Freund complete adjuvant which was given three times completely protected an Aotus lemurinus azure (karotype VI) monkey against homologous challenge; however, immunization with the same polypeptides with a muramyl dipeptide derivative [MDP-Lys(L18)] did not protect a second Aotus monkey, even though comparable high antibody titers were induced.     

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.     

Synthetic peptides as antigens: pitfalls of conjugation methods

Peptide-carrier conjugates were prepared using 9 different synthetic peptides, 3 carrier proteins and 4 coupling reagents. Residues of the carrier protein that were modified by different coupling reagents (e.g., glutaraldehyde, carbodiimides, bis-diazotized benzidine) were found to elicit specific antibodies that reacted with unrelated carrier proteins treated with the same coupling agent. To demonstrate the presence of peptide antibodies in an antiserum raised against a peptide-carrier conjugate, it was necessary to use a antigen the peptide coupled to another carrier by means of a different coupling agent. Some of the commonly used conjugation methods were found to lead to conjugates of insufficient stability and sometimes also altered the antigenic properties of the peptide moiety. These difficulties can be overcome by additional control experiments designed to test the quality and the peptide-carrier conjugates.     

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.     

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.     

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.     

Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites

The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.     

Plasmodium falciparum andP. berghei: detection of sporozoites and the circumsporozoite proteins in the saliva ofAnopheles stephensi mosquitoes

Sporozoites and free circumsporozoite (CS) protein were stained immunoenzymatically in 1-min saliva samples collected fromAnopheles stephensi mosquitoes infected with eitherPlasmodium berghei orP. falciparum. The number of sporozoites in 1-min saliva-streak samples significantly increased as the salivary gland index rose from 3+ to 4+. ForP. berghei-infected mosquitoes from which saliva had been collected before 30 days postfeed, the median sporozoite counts for 3+ and 4+ gland indexes were 4.5 and 116, respectively. ForP. falciparum-infected mosquitoes, the median counts obtained in two experiments were 4.5 and 14.5 (3+) and 97 and 107 (4+), respectively. The frequency of sporozoite detection in the saliva of mosquitoes containing <100 salivary-gland sporozoites was low (0.1), whereas that in the saliva of mosquitoes with >100 sporozoites was high (0.96). In highly infected 4+P. berghei-infected mosquitoes from which saliva had been collected after 30 days postinfection, both the volume of saliva collected and the median number of sporozoites recovered decreased significantly.     

Multiplex assay for simultaneous measurement of antibodies to multiple Plasmodium falciparum antigens

Antibodies to Plasmodium falciparum are classically measured using the enzyme-linked immunosorbent assay (ELISA). Although highly sensitive, this technique is labor-intensive when large numbers of samples must be screened against multiple antigens. The suspension array technology (SAT) might be an alterative to ELISA, as it allows measurement of antibodies against multiple antigens simultaneously with a small volume of sample. This study sought to adapt the new SAT multiplex system for measuring antibodies against nine malarial vaccine candidate antigens, including recombinant proteins from two variants of merozoite surface protein 1, two variants of apical merozoite antigen 1, erythrocyte binding antigen 175, merozoite surface protein 3, and peptides from the circumsporozoite protein, ring erythrocyte surface antigen, and liver-stage antigen 1. Various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Cameroonian adults were screened by SAT in mono- and multiplex formats and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10- to 100-fold less than that needed for ELISA. Excellent agreement was found between the single and multiplex formats (R > or = 0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 mul of plasma, and had a good correlation with ELISA. Thus, SAT provides an important new tool for studying the immune response to malaria rapidly and efficiently in large populations, even when the amount of plasma available is limited, e.g., in studies of neonates or finger-prick blood.     

Malaria-induced acquisition of antibodies to Plasmodium falciparum variant surface antigens

In areas of intense Plasmodium falciparum transmission, protective immunity is acquired during childhood in parallel with acquisition of agglutinating antibodies to parasite-encoded variant surface antigens (VSA) expressed on parasitized red blood cells. In a semi-immune child in such an area, clinical disease is caused mainly by parasites expressing VSA not recognized by preexisting VSA-specific antibodies in that child. Such malaria episodes are known to cause an increase in agglutinating antibodies specifically recognizing VSA expressed by the parasite isolate causing the illness, whereas antibody responses to other parasite isolates are relatively unaffected. However, the detailed kinetics of this VSA antibody acquisition are unknown and hence were the aim of this study. We show that P. falciparum malaria in Ghanaian children generally caused a rapid and sustained increase in variant-specific VSA antibody levels, while more transient and limited increases in levels of antibodies to VSA expressed by other parasite isolates were also seen. Plasma VSA antibody levels were positively correlated with the age of the healthy plasma donors but negatively correlated with the age of the parasite donors (the malaria patient). The data from this first detailed longitudinal study of acquisition of VSA antibodies support the hypothesis that naturally acquired protective immunity to P. falciparum malaria is mediated, at least in part, by VSA-specific antibodies.     

Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice

Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a compound that significantly affects blood stage parasitemia and transmission of murine malaria and also inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development. Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporozoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.     

Glycolipid anchorage of Plasmodium falciparum surface antigens.

Human red blood cells (RBC) were infected with the malarial parasite Plasmodium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine-labelled protein into the supernatant after incubation with phosphatidylinositol-specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methionine-labelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins.     

Identification of plasma membrane proteins involved in the hepatocyte invasion of Plasmodium falciparum sporozoites

To determine whether surface proteins of hepatocytes might be involved in the sporozoite invasion, plasma membrane proteins were prepared from human livers with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) and radiolabelled with 125I (Iodogen; 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril). The labelled proteins were incubated with Plasmodium falciparum sporozoites and cross-linked with DSP (dithio-bis-succinimidylpropionate). Radiolabelled proteins released by reduction after repeated washing of the sporozoite-complex were separated by SDS-PAGE and autoradiographed. Two human hepatocyte membrane proteins of 20 and 55 kDa were found to be involved in the initial binding of P. falciparum sporozoites. The electrophoretically purified 20- and 55-kDa proteins both inhibited the binding of the corresponding radiolabelled proteins to P. falciparum sporozoites and reduced the invasion of sporozoites in an in vitro assay. We propose that these 20-kDa and 55-kDa proteins represent putative human hepatocyte receptors for P. falciparum sporozoite invasion.     

Variation in the precursor to the major merozoite surface antigens of Plasmodium falciparum

The major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight polypeptide. The precursor to the major merozoite surface antigen (PMMSA) has conserved and variable antigenic determinants and varies in size in different isolates. Since the protein is a candidate for a malaria vaccine, it is important to understand the molecular basis of this variation. We present the complete sequence of the PMMSA of the Papua New Guinea isolate FC27 and the partial sequence of the West African isolate NF7. The gene consists of blocks of sequence which are either conserved or variable between different isolates. Variable sequences fall into two distinct types, indicating that the PMMSA is encoded by dimorphic alleles that undergo recombination within conserved blocks at the 5' end of the gene. Genetic exchange is not apparent within the other two-thirds of the gene in 12 isolates, suggesting strong selection against such recombinants. The most variable block located near the 5' end contains repeats that are different in independent cloned isolates. This variation presumably accounts for much of the size and antigenic variability.