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.     

High frequency of antibody response to Plasmodium falciparum gametocyte antigens during acute malaria infections in Papua New Guinea highlanders.

Sera from 62 adult Papua New Guinea highlanders with suspected acute malaria were tested by competitive ELISA for the presence of antibodies capable of inhibiting binding of 8 monoclonal antibodies (Mabs) directed against epitopes on gametocytes of Plasmodium falciparum. Between 33% and 72% of the malaria cases were inhibitory, depending on the Mab. There was no difference between the proportion of persons with P. falciparum (asexuals or gametocytes) and P. vivax whose sera inhibited Mab binding, but all 3 categories had a significantly higher proportion of inhibitors than persons who were malaria negative. The amount of gametocyte antibody recognizing epitopes on Pfs 48/45 and Pfs 230 increased with increasing numbers of previous malaria episodes. The proportion of sera from these relatively nonimmune adults which had gamete antibodies was similar to the proportion seen in sera from a highly endemic area, suggesting that antibody responses to these epitopes are a part of the initial response observed after a limited number of malaria episodes.     

Antibodies to Plasmodium falciparum gamete surface antigens in Papua New Guinea sera

Sera from individuals living in malaria endemic areas of Papua New Guinea were tested for their effect on infectivity of Plasmodium falciparum gametocytes grown in culture to Anopheles freeborni mosquitoes. Consistent reduction of infectivity to less than 5% of control was observed with nine out of the 41 sera from the endemic area tested and also with three out of seven sera tested from individuals rarely exposed to malaria infection. Gamete surface antigens recognized by the sera were investigated by immunoprecipitation from 125I surface-labelled gametes extracted in SDS and Triton X-100. The main antigens recognized were of the same mol. wt (230, 48 and 45 kD) as those known to be targets of transmission-blocking monoclonal antibodies. A significant negative correlation was observed between the total ct/min immunoprecipitated from surface-labelled gametes by the sera and the average number of oocysts per gut observed in membrane feeding experiments with these sera. Spearmann's rank correlation coefficient indicated that suppression of infectivity correlated strongly with the presence of antibodies against the 230 kD protein; there was no significant correlation between suppression and antibodies to the 48/45 kD proteins. The antibody response to the different gamete surface antigens varied greatly in sera from the endemic areas suggesting that individuals respond differently to each gamete antigen.     

Human antibody responses to epitopes on the Plasmodium falciparum gametocyte antigen PFS 48/45 and their relationship to infectivity of gametocyte carriers.

Antibodies in human sera recognizing epitopes I, IIa, III, and IV on the Plasmodium falciparum gametocyte antigen Pfs 48/45 have been investigated by competitive enzyme-linked immunosorbent assay. More than one-third of the residents of three villages in Madang, Papua New Guinea responded to epitopes I, IIa and III, with little variation by village or with time. There was a bimodal distribution of positive sera by age, with the highest proportion of responders in the 5-9- and greater than 20-year-old age groups. The data suggest a lower prevalence of antibodies against epitopes IIa and III in P. falciparum gametocyte carriers than in non-carriers. Enhancement of binding of monoclonal antibodies to epitopes IIa and III was also observed more frequently with sera from gametocyte carriers. Sera from gametocyte carriers in Papua New Guinea and Thailand, whose infectivity to mosquitoes had been tested, were used to examine the relationship between recognition of particular epitopes and infectivity. There was a significant association between lack of infectivity of P. falciparum gametocyte carriers and recognition of epitope IIa on Pfs 48/45 by antibodies in their sera.     

Immunogenicity of malaria transmission-blocking vaccine candidate, y230.CA14 following crosslinking in the presence of tetanus toxoid

Proteolytically processed 310 kDa form of Plasmodium falciparum gamete surface antigen, Pfs230, is the target of malaria transmission-blocking monoclonal antibodies. To design a recombinant malaria transmission-blocking subunit vaccine, the amino terminus of the 310 kDa surface-exposed form of Pfs230 was mapped to amino acids (aa) 522 and 584 using a series of peptides and recombinant proteins encoding distinct regions of Pfs230. Antiserum generated against an Escherichia coli-produced recombinant protein, spanning the Pfs230 processing site and extending into the cysteine domains, r230/MBP.C (aa 443-1132), reduced parasite infectivity by 71.2-89.8%. To determine if the region spanning the cleavage site blocked malaria transmission when produced as a secreted protein by Saccharomyces cerevisiae, y230.CA14 (aa 467-584) was generated, purified, emulsified in adjuvant and used to vaccinate mice. In contrast to E. coli-produced r230/MBP.C, the immune response generated against y230. CA14 was very weak. To enhance the response, y230.CA14 was mixed with tetanus toxoid, chemically crosslinked, repurifed, and its immunogenicty compared with unconjugated y230.CA14. Conjugated-y230. CA14/TT required fewer booster injections to induce an immune response against Pfs230 and the antibodies generated reacted with the surface of intact gametes and immunoprecipitated radiolabelled Pfs230 extracted from 125I surface-labelled gametes to a greater extent. After seven injections, all y230.CA14 vaccinated mice developed anti-Pfs230 antibodies and the isotype profile was the same. In addition to enhancing the initial immune response generated against y230.CA14, conjugation focuses the immune response toward epitopes within the region of Pfs230 present on the surface of the gamete.     

Diversity of antigens expressed on the surface of erythrocytes infected with mature Plasmodium falciparum parasites in Papua New Guinea

Antigens were detected on the surface of erythrocytes from children with acute falciparum malaria in Madang, Papua New Guinea. These parasite-induced erythrocyte surface antigens (PIESA) were serotyped with convalescent sera from children and hyperimmune sera from adults in parasite infected cell agglutination assays (PICAs) and by inhibition of binding of infected cells to melanoma cells. Extensive serological diversity of PIESA was demonstrated. A significant correlation between serotypes defined by reactivity of immune sera in PICA and inhibition of melanoma cell binding (MCB) was observed. This suggests that both assays measure antibody responses to the same antigen(s). Increased recognition of different PIESA specificities with age is consistent with the hypothesis that repeated exposure to malaria confers immunity against a range of PIESA serotypes and parallels the development of clinical immunity to malaria in this area of Papua New Guinea.     

Rapid onset of transmission-reducing antibodies in javanese migrants exposed to malaria in papua, indonesia

Transmission of Plasmodium falciparum malaria is initiated by sexual stages in the mosquito. Anti-Pfs48/45 and anti-Pfs230 sexual stage antibodies that are ingested together with parasites can reduce parasite development and subsequently malaria transmission. Acquisition of sexual stage immunity was studied in a cohort of 102 non-immune Javanese individuals migrating to hyperendemic Papua Indonesia. Seroprevalence of antibodies against Pfs48/45 and Pfs230 and functional transmission-reducing activity (TRA) were measured upon arrival and at 6, 12, and 24 months. Asexual parasitemia and gametocytemia were assessed every two weeks. The TRA and seroreactivity increased with the number of P. falciparum infections. The longitudinally sustained association between TRA and antibodies against Pfs48/45 (odds ratio [OR] = 3.74, 95% confidence interval [CI] = 1.51-9.29) and Pfs230 (OR = 3.72, 95% CI = 1.36-10.17) suggests that functional transmission reducing immunity is acquired after limited exposure to infection.     

Antibody responses to Plasmodium falciparum gametocyte antigens during and after malaria attacks in schoolchildren from Madang, Papua New Guinea

Sera from 49 school children in Madang, Papua New Guinea with malaria and follow-up sera from 40 of these cases were tested by competitive ELISA for antibodies capable of inhibiting binding of eight monoclonal antibodies (MoAbs) to Plasmodium falciparum gametocytes. The proportion of sera inhibiting each MoAb ranged from 31.2% to 85.7%. At follow-up, the proportion of inhibitory sera decreased for 3 MoAbs, did not change significantly for 4 MoAbs and increased for one MoAb. When sera were grouped according to whether the follow-up blood slide was positive or negative, further trends emerged for MoAbs against the gamete surface antigen Pfs 48/45. Antibody levels to the IA3-B8 epitope decreased in follow-up positive cases, but remained unchanged for follow-up negative cases. The converse was observed for the IIC5-B10 epitope with an increase of antibody in follow-up positive cases and no change in the negative cases. Amount of antibody to the 3G12/58 epitope decreased when the follow-up was negative but not when it was positive. Increase in antibody to the 3E12/58 epitope occurred at the follow-up sample irrespective of the blood slide result. Thus four distinct patterns of longitudinal antibody response were observed against four epitopes on the same molecule.     

Conserved and variant epitopes of target antigens of transmission-blocking antibodies among isolates of Plasmodium falciparum from Malaysia.

Monoclonal antibodies (MAbs) directed against different epitope regions on three sexual stage-specific gamete surface proteins of Plasmodium falciparum, Pfs 25, Pfs 230, and Pfs 48/45, were used to study the genetic diversity of these epitopes among fresh isolates of P. falciparum from Malaysia, using immunofluorescence microscopy (IFA). Among 45 Malaysian isolates, one epitope of Pfs 25, designated region I, showed evidence of variable reactivity with MAbs among different isolates; the Pfs 25 epitope, region II, was universally recognized by MAbs in all isolates. Two apparently distinct epitope regions of Pfs 230 were defined by MAbs, one of which was universally recognized by MAbs among the 45 isolates; the other was conserved in all but three isolates. The epitope regions of gamete-surface protein Pfs 48/45, designated regions I, IIa, IIb, IIc, III, and IV, were examined for reactivity by IFA in 33 isolates. Epitope regions I, IIb, III, and IV were conserved in all isolates; regions IIa and IIc existed in variant forms.     

Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement-fixing

We have studied the properties of 16 newly derived monoclonal antibodies (MoAbs) against Pfs230, a gamete surface protein of Plasmodium falciparum and a target of transmission-blocking antibodies. All 16 MoAbs recognized Pfs230 by immunoprecipitation from non-ionic detergent extracts of the protein radio-labelled with ¹²⁵Iodine. The MoAbs also recognized this protein on Western blots under non-reducing conditions but none of them recognized the protein under reducing conditions. Using an immunoradiometric assay the MoAbs appear to define nine different epitope regions. The MoAbs were tested for their ability to lyse extra-cellular female gametes of P. falciparum isolate 3D7. Eight of the MoAbs, all of isotype IgG2a, mediated complement-dependent lysis of the gametes; seven of the MoAbs, all isotype IgG1, failed to lyse the gametes in the presence of active complement. The eight complement-fixing MoAbs mediated almost total suppression of infectivity of gameto-cytes of P. falciparum 3D7 to mosquitoes; where tested this suppression was mainly complement-dependent. The seven non-complement-fixing MoAbs had no significant effect on the infectivity of gametocytes of P. falciparum 3D7 to mosquitoes. When tested by immunofluorescence the target epitopes of all the MoAbs were conserved in each of the five different isolates of P. falciparum which were tested.     

Assessment of the role of the humoral response to Plasmodium falciparum MSP2 compared to RESA and SPf66 in protecting Papua New Guinean children from clinical malaria

The prevalence and concentration of naturally acquired humoral response (IgG) to merozoite surface protein 2 (MSP2), RESA, SPf66 and crude schizont extract were measured in a population living in a malaria highly endemic area of Papua New Guinea. A prospective longitudinal study in 0–5–15 year old children was conducted for one year in order to examine the relationship between the humoral response to these antigens and subsequent susceptibility to clinical malaria using a series of clinical definitions. The prevalence and concentration of antibodies to all antigens increased with age. Such correlation with age was most marked for MSP2 recombinant proteins. When age and previous exposure were controlled for, only antibody levels to MSP2 recombinant proteins (3D7 andd3D7) and to RESA predicted a reduction in incidence rate of episodes of clinical malaria. Our results support the inclusion of the recombinant proteins of the 3D7 allelic family of merozoite surface antigen 2 and RESA into a subunit vaccine against malaria.,     

Anti-gamete antibodies block transmission of human vivax malaria to mosquitoes

Antibodies were raised in rabbits by immunizing against fresh unfixed or cryopreserved female gametes of the human malaria pathogen Plasmodium vivax. The antibodies were shown to react with the surface of gametes by the indirect immunofluorescent test. When parasite isolates from P. vivax infected individuals were fed through a membrane to Anopheles tessellatus mosquitoes in the presence of immune rabbit sera, they completely blocked the infectivity of the parasite isolates to the vector. Immunoglobulins separated from these sera also blocked infectivity to the same extent as did the immune sera indicating that antibodies were responsible for the transmission blocking effect of the sera. This study indicated that P. vivax like other malaria parasites is highly susceptible to anti gamete transmission blocking immunity.     

Human immune recognition of recombinant proteins representing discrete domains of the Plasmodium falciparum gamete surface protein, Pfs230

The 230 kD gamelocyte/gamete-specific surface protein of Plasmodium falciparum, Pfs230, is a target of antibodies which inhibit the development of the parasite inside the mosquito vector. A transmission blocking vaccine based on Pfs230 may be a powerful tool for malaria control. As a first step, Pfs230 has been expressed in E. coli as a series of recombinant proteins, fused to maltose binding protein. We have used the fusion proteins to assess cellular and humoral immune responses to Pfs230 in malaria-immune adult Gambian blood donors; responses to the fusion proteins have been compared with responses to native Pfs230. The tetrapeptide repeat region of the molecule appears to be immunodominant for both antibody-producing cells and peripheral blood T cells. We postulate that this may represent a mechanism for immune evasion since the N-terminal repeat region of the molecule is cleaved from the mature protein and shed into the plasma. Responses to fusion proteins representing the seven-cysteine motifs were correlated within individual donors, suggesting that cross-reactive epitopes occur within the motifs. Antibody responses to recombinant proteins were poorly correlated with responses to native Pfs230 suggesting that dominant epitopes of the native protein are not adequately represented in the recombinant proteins. Although prokaryotic expression products may be suitable for induction of cellular immune responses to Pfs230, alternative expression systems may be needed for creation of appropriate B cell epitopes.     

High Levels of Genetic Diversity of Plasmodium falciparum Populations in Papua New Guinea despite Variable Infection Prevalence

High levels of genetic diversity in Plasmodium falciparum populations are an obstacle to malaria control. Here, we investigate the relationship between local variation in malaria epidemiology and parasite genetic diversity in Papua New Guinea (PNG). Cross-sectional malaria surveys were performed in 14 villages spanning four distinct malaria-endemic areas on the north coast, including one area that was sampled during the dry season. High-resolution msp2 genotyping of 2,147 blood samples identified 761 P. falciparum infections containing a total of 1,392 clones whose genotypes were used to measure genetic diversity. Considerable variability in infection prevalence and mean multiplicity of infection was observed at all of the study sites, with the area sampled during the dry season showing particularly striking local variability. Genetic diversity was strongly associated with multiplicity of infection but not with infection prevalence. In highly endemic areas, differences in infection prevalence may not translate into a decrease in parasite population diversity.     

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.     

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Summary Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.     

Seroepidemiology of malaria in northern Thailand: II. Focal and temporal variations in endemicity

Four population groups from regions of Northern Thailand were surveyed for the presence of antibodies to Plasmodium falciparum using the indirect immunofluorescent antibody (IFA) test. Each of the four populations was selected from areas known to represent different patterns of malaria transmission. Group 1 was from an area where there had been no malaria transmission for approximately 30 years. Individuals in this group below age 40 showed an extremely low prevalence of malaria antibodies. Group 2 was chosen from an area where low levels of transmission have continued despite more than 30 years of DDT spraying. In this group the age related pattern of malaria antibodies varied from village to village but in all villages there was a sharp increase in the prevalence of IFA positive tests in individuals over 30. The third group has had continuously high levels of transmission. Although there are differences in the age related prevalence of IFA positives when individual villages are compared, there is a greater prevalence at all ages than in the first two groups. The fourth group was selected from an area where transmission had recently resumed after freedom from indigenous cases for approximately six years. There was little difference in the prevalence of IFA positive individuals below the age of 25 but above that there was a steady increase in prevalence with age. The correlation of IFA antibody positives with known patterns of malaria transmission in these four areas demonstrates the usefulness of this serological technique in assessing malaria endemicity and the effectiveness of control measures as well as in the interpretation of other malaria statistics.     

Humoral immune responses in Gambians to Pfs16, an immunodominant, Plasmodium falciparum integral membrane protein

Naturally acquired humoral immune responses to Pfs16, an integral membrane protein expressed in Plasmodium falciparum gametocytes and sporozoites, were investigated in The Gambia. A high prevalence of antibodies to this molecule was detected by peptide ELISA. Ninety-three per cent of the people taking part in a survey at the end of the rainy season (November) had serum antibodies to one or more synthetic peptides spanning the sequence; 88% reacted with one particular peptide sequence (IMLIILSGIVGFKVK) whereas only one out of ten non-Gambians (taking anti-malarial prophylaxis with no history of infection) reacted with the peptide. Epitope mapping with mouse MoAbs has shown that this peptide is located on a part of the molecule differing from immunodominant regions of the molecule identified in a previous study in Papua New Guinea.     

A longitudinal study of immune responses to Plasmodium falciparum sexual stage antigens in Tanzanian adults

Next to children, adults form a substantial part of the infectious reservoir that is responsible for the spread of malaria. In this longitudinal study, we determined sexual stage immune responses to Plasmodium falciparum and infectiousness to mosquitoes in adults from an area with intense malaria transmission. A cohort of 43 Tanzanian adults was followed for 18 months. Parasitological data were collected monthly; serum once every three months. Antibody prevalences were determined for sexual stage antigens Pfs230 and Pfs48/45 and circumsporozoite protein (NANP5)(n = 199). Functional transmission reducing activity (TRA) was assessed by standard membrane feeding assay (SMFA; n = 85). Cumulative parasite prevalence was 67.4% (29/43) for asexual stages and 34.9% (15/43) for gametocytes. Enrolment antibody prevalence was 95.3% (41/43) for NANP5, 18.9% (7/37) for Pfs230 and 7% (3/43) for Pfs48/45 epitope 3. TRA > 50% reduction was seen in 48.2% (41/85) and TRA > 90% reduction in 4.7% (4/85) of the samples. Our findings of low and inconsistent sexual stage immune responses are likely to be the result of a low exposure to gametocytes in this older age group. This may in turn be caused by effective asexual stage immunity. We conclude that the infectivity of older individuals is less likely to be affected by sexual stage immunity.     

Immunoprecipitation of biosynthetically-labelled proteins from different Papua New Guinea Plasmodium falciparum isolates by sera from individuals in the endemic area

The human serum antibody response to Plasmodium falciparum infection in Papua New Guinea has been studied by electrophoretic analysis of immunoprecipitated biosynthetically-labelled malaria proteins from three different isolates maintained in long-term in vitro culture. Differences in protein antigenic composition in different lines have been described and simplified by examination of antigens recognized only by hyperimmune serum. An in vitro assay has been used to screen various human sera containing antimalarial antibody for their ability to inhibit parasite growth and the immunoprecipitation profiles of non-inhibitory sera have been compared with those of a hyperimmune serum pool. In the discussion, emphasis is placed on the value of immunoprecipitation analyses using clinically-defined sera with known in vitro function in the identification of antigens which may be responsible for the induction of host-protective immunity.