A longitudinal study of human antibody responses to Plasmodium falciparum rhoptry-associated protein 1 in a region of seasonal and unstable malaria transmission

Abstract

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that of P. falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed to P. falciparum malaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies detectable with at least one rRAP1 protein. However, the anti-RAP1 antibody responses were detected only during or shortly after clinical malarial infections. RAP1 antibody levels declined rapidly (within 1 to 2 months) following drug treatment of the infections. No anti-RAP1 antibodies were usually detected a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of individuals after clinical malarial infections. For some individuals who experienced more than one malarial infection, a higher anti-RAP1 antibody response to subsequent infections than to earlier infections was observed. This suggested secondary responses to RAP1 and thus the development of immunological memory for RAP1.

FIG. 1

Schematic representation showing mature P. falciparum RAP1 and rRAP1 proteins. P2 to P5 are proteins fused to hexahistidine tag (■); C1 and C2 are fused to GST (). , region of repeats related to the KSSSPS motif (between amino acid 123 and 164). ↑, cleavage site (between amino acid 190 and 191) of mature RAP1 to yield p65 fragment (35). , inhibitory monoclonal antibody epitope (L₂₀₂ TPLEELYP₂₁₀ [16]). , cysteine-rich region (between amino acid 353 and 616). Amino acid numbering is as in the work of Ridley et al. (37). This figure has been modified from the work of Fonjungo et al. (13).

FIG. 2

Reactivity profiles of IgG antibodies detectable with rRAP1 proteins C1 (a), P3 (b), and P5 (c) in 499 serum samples donated by 53 individuals between 1991 and 1995. Each serum sample was tested by ELISA at a dilution of 1:500, and reactions were developed with peroxidase-conjugated rabbit anti-human IgG diluted 1:5,000. Cutoff levels (means + 2 standard deviations of 33 European serum samples) and the indicated range of OD values above the cutoffs were used to grade arbitrarily the strength of each reaction. □, below cutoff level; ▧, above cutoff (OD < 0.5); ▥, above cutoff (OD = 0.5 to 1.0); ■, above cutoff (OD > 1.0); #, clinical malaria infection with no available plasma sample; Conv, convalescence sample collected 30 to 40 days after clinical malaria infection.

FIG. 2

Reactivity profiles of IgG antibodies detectable with rRAP1 proteins C1 (a), P3 (b), and P5 (c) in 499 serum samples donated by 53 individuals between 1991 and 1995. Each serum sample was tested by ELISA at a dilution of 1:500, and reactions were developed with peroxidase-conjugated rabbit anti-human IgG diluted 1:5,000. Cutoff levels (means + 2 standard deviations of 33 European serum samples) and the indicated range of OD values above the cutoffs were used to grade arbitrarily the strength of each reaction. □, below cutoff level; ▧, above cutoff (OD < 0.5); ▥, above cutoff (OD = 0.5 to 1.0); ■, above cutoff (OD > 1.0); #, clinical malaria infection with no available plasma sample; Conv, convalescence sample collected 30 to 40 days after clinical malaria infection.

FIG. 2

Reactivity profiles of IgG antibodies detectable with rRAP1 proteins C1 (a), P3 (b), and P5 (c) in 499 serum samples donated by 53 individuals between 1991 and 1995. Each serum sample was tested by ELISA at a dilution of 1:500, and reactions were developed with peroxidase-conjugated rabbit anti-human IgG diluted 1:5,000. Cutoff levels (means + 2 standard deviations of 33 European serum samples) and the indicated range of OD values above the cutoffs were used to grade arbitrarily the strength of each reaction. □, below cutoff level; ▧, above cutoff (OD < 0.5); ▥, above cutoff (OD = 0.5 to 1.0); ■, above cutoff (OD > 1.0); #, clinical malaria infection with no available plasma sample; Conv, convalescence sample collected 30 to 40 days after clinical malaria infection.

FIG. 3

Different patterns of IgG antibody responses to RAP1 in natural P. falciparum infections. Each panel illustrates the time course of response(s) of one individual as detected by ELISA with six rRAP1 proteins. The x axes show the month and the year when each test sample was collected. Dates of samples collected during documented episodes of clinical malaria are underlined. PCR results, also along the x axes, indicate the absence (−) or presence (+) of P. falciparum in blood samples of each individual. nd, PCR was not done.

FIG. 3

Different patterns of IgG antibody responses to RAP1 in natural P. falciparum infections. Each panel illustrates the time course of response(s) of one individual as detected by ELISA with six rRAP1 proteins. The x axes show the month and the year when each test sample was collected. Dates of samples collected during documented episodes of clinical malaria are underlined. PCR results, also along the x axes, indicate the absence (−) or presence (+) of P. falciparum in blood samples of each individual. nd, PCR was not done.

FIG. 4

IgG responses to RAP1 are short-lived after recovery from P. falciparum infection. Reactivities of rRAP1 antigens with IgG in sera donated by five different individuals on the indicated dates before, during, and after drug-cured clinical malarial episodes (asterisks) are shown. Note a rapid decline of antibody levels in all cases. A cutoff value indicated by a horizontal line is that of P4; cutoffs for the other antigens were lower (see Materials and Methods).