Does Antibody Avidity to Plasmodium falciparum Merozoite Antigens Increase with Age in Individuals Living in Malaria-Endemic Areas?

Abstract

High-avidity antibodies (Abs) are acquired after a few Plasmodium falciparum infections in low transmission areas, but it remains unclear if Ab avidity to different merozoite antigens increases with age in individuals with persistent antigenemia and, if so, when a fully mature Ab response occurs. The study used plasma samples collected between 1996 and 1998 from 566 individuals aged 4 to 84 years in Simbok, Cameroon, where residents received an estimated 1.6 infectious mosquito bites/person/night. Plasma samples were examined for Ab levels (median fluorescence intensity [MFI]) and Ab avidity index (AI) (where AI = [MFI after treatment with 2 M NH₄SCN/MFI without salt] × 100) using a bead-based multiplex immunoassay for recombinant AMA1, EBA-175, MSP1-42 (3D7, FVO), MSP2 (3D7, Fc27), and MSP3. Blood-smear positivity for P. falciparum declined with age from 54.3% at 4 to 5 years to 18% at 16 to 40 years and <11% at >40 years of age, although most individuals had submicroscopic parasitemia. Ab affinity maturation, based on age-related patterns of median AI, percentage of individuals with AI of ≥50, and strength of association between MFI and AI, occurred at different rates among the antigens; they developed rapidly before age 4 years for AMA1, increased gradually with age for EBA-175 and MSP1 until ∼16 to 25 years, but occurred negligibly for MSP2 and MSP3. In a hyperendemic area with perennial transmission, affinity maturation resulting in an increase in the proportion of high-avidity Abs occurred for some merozoite antigens, in parallel with a decline in malaria slide passivity, but not for others.

Keywords: antibody avidity; malaria; merozoite antigens.

FIG 1

Malaria prevalence in Simbok. Results for slide-positivity are based on thick films for all 566 individuals. PCR was performed using primers for 18S rRNA and Cox3 and results are based on 150 samples randomly selected from all age groups. The age distribution of the 150 samples used for PCR was as follows: 4 to 5 years (n = 8); 6 to 10 years (n = 21); 11 to 15 years (n = 29); 16 to 20 years (n = 11); 21 to 25 years (n = 13); 26 to 30 years (n = 13); 31 to 40 years (n = 19); 41 to 50 years (n = 13); and 51+ years (n = 23). Data labeled Cox3 + 18S shows the percentage of samples that were PCR-positive with one or both primers.

FIG 2

Change in Ab levels with increasing age. Ab levels (MFI) for each of the 566 individuals enrolled in the study were divided into 9 age groups. (A to G) Box plots represent the 25^th, 50^th, and 75^th percentiles (horizontal lines of box) and minimum/maximum values (tip of whiskers) for each age group. Sample sizes are provided in Table 1. Median Ab levels were compared among all age groups using the Kruskal-Wallis (KW) test. (H) Pairwise MFI comparisons (Wilcoxon rank sum test) between the age group with observed peak median MFI (31 to 40 years) and other age groups for EBA-175 and MSP1-42 3D7 and FVO. The results, after Bonferroni adjustment of P values, indicate that the increase in Ab levels with age actually became maximal at 11 to 15 years for EBA-175 and 16 to 20 years for MSP1-42 (both alleles), and did not significantly change thereafter. NA, not applicable.

FIG 3

Change in avidity index (AI) with age. The AI (% Ab remaining bound after treatment with 2 M NH₄SCN) was calculated for each individual who was Ab positive, i.e., with MFI value of ≥500 or >mean + 3SD of malaria-naive U.S. controls (whichever was higher). (A to G) Box plots represent the 25^th, 50^th, and 75^th percentiles (horizontal lines of box) and minimum/maximum values (tip of whiskers) for each age group. Sample sizes are provided in Table 1. Median AI was compared among all age groups by way of the Kruskal-Wallis (KW) test. (H) Pairwise AI comparisons (for antigens with significant KW) between age groups with observed peak median AI (73% at 26 to 30 years for EBA-175, 68% at 31 to 40 years for MSP1 3D7, and 78% at 31 to 40 years for MSP1 FVO) and other ordered age groups. The P values were adjusted for multiple comparisons using the Bonferroni method. The results indicate that the increase in AI with age actually became maximal at 16 to 20 years and 21 to 25 years for MSP1-42 and EBA-175, respectively, and stable thereafter.

FIG 4

Spearman correlations (r_s) and 95% confidence intervals between MFI and AI. Spearman r_s coefficients with 95% confidence intervals (CIs) for the monotonic relationship between antibody levels expressed as median fluorescence intensity (MFI) and antibody avidity expressed as avidity index (AI) are represented for each antigen. The results are shown for antibody-positive individuals combined into larger age groups (4 to 10 years, 11 to 20 years, 21 to 30 years, and 31+ years) to reduce complexity and increase power of the correlation analysis. The strength of the monotonic relationship between MFI and AI was indicated as weak (0.000<|r_s|<0.400), moderate (0.400<|r_s|<0.700), or strong (0.700<|r_s|<1.000).

FIG 5

The percentage of individuals with >50% high-avidity antibodies. The percentage of Ab-positive individuals with AI ≥50% was determined for each age group and for each antigen.

FIG 6

Comparison of antibody response to AMA1 between infants 12 months of age and adults. (A and B) Plasma samples from 38 adults and 55 infants living in Ngali II village with equally high malaria transmission as in Simbok village were compared for avidity index (AI) (A) and the monotonic relationship between MFI and AI for Ab-positive participants (B). The 38 adults were of reproductive age and consisted of 14 males, 9 nonpregnant females, and 15 pregnant women. Note that 1.5 M NH₄SCN was used in this part of the study, since it provided a wider range of AI in infants (19). (A) Box plots represent the 25^th, 50^th, and 75^th percentiles (horizontal lines of box) and minimum/maximum values (tip of whiskers). (B) Horizontal lines show the Spearman correlation coefficients (dot) and lower/upper limits of 95% confidence interval.

FIG 7

Summary of affinity maturation patterns for antibodies to P. falciparum merozoite antigens in an area of high malaria transmission intensity. The antibody avidity index (AI) to 5 merozoite antigens was measured for 566 individuals aged 4 to 84 years who lived in the rural village of Simbok, Cameroon, where they were exposed to infectious mosquito bites daily. In addition, AMA1 AI was measured in 38 adults and 55 1-year-old infants living in the nearby village of Ngali II with similar malaria transmission intensity. In general, three distinct AI-versus-age patterns were observed based on various parameters, including median AI, percentage of the population with ≥50% high-avidity antibodies, relationship between AI and antibody levels, and interplay with antiparasite immunity.