Naturally acquired immunity to Plasmodium Falciparum Antigens in Children less than four Years of age in a Malaria Holoendemic Region of Western Kenya

Abstract

The mechanism of age-dependent acquisition of immunity to P. Jalciparum in malaria endemic areas is less clear. Understanding how immunity is acquired with age in humans requires a rational design and development of methodology for evaluating the key features of naturally acquired immunity especially in infants and young children. However, immunological studies in infants and young children have been hampered by the ethical requirement that only small blood volumes can be drawn for-the studies. To circumvent this problem, this study was designed to use finger prick blood samples to investigate the level and age dependent development of immunological responses to Plasmodium Jalciparum antigens in children less than 4 years of age from western Kenya. A total of 110 individuals were enrolled and stratified into four age groups: 0-12 months, 13-24 months, 25-36 months, and 37-48 months. 0.5-lml of finger prick blood was collected from children and separated by Ficol-Hypaque density gradient centrifugation to obtain the peripheral blood mononuclear cells (PBMC) and the plasma. Varying concentrations of PBMC (1x105, 2x105, and 5x105) were plated per well and cultured for 72 and 120 hours with each mitogen (phytohaemoglutinin [PHA], and phorbal 12 meristate 13 acetate-ionomycin [PMA-I]) and Plasmodium Jalciparum antigenic peptides (Liver stage antigen-l [LSA-l] and Merozoite surface protein-l [MSP-l]) in humidified incubator at 37.2°C and 5% carbon dioxide in cRPMI 1640 media. After incubation, the supernatant from each well was tested for the presence of interleukin 10 (IL-I0) and interferon gamma (IFN-y) cytokines and plasma was used to test for the presence of total immunoglobulin M (IgM), IgG and IgG subclass antibodies to Plasmodium Jalciparum malaria antigens by enzyme linked immunosorbent assays (ELISAs). xv The results showed that the frequencies of IFN-y responses to both antigens showed an increasing trend while the frequencies of IL-I 0 were similar across the age groups. The frequencies and levels of total IgG, IgM and IgG subclass responses to pre erythrocytic and erythrocytic antigens were variable. For IgG subclasses, only IgG 1 and IgG3 but not IgG2 and IgG4 to all antigens were found at detectable levels. Total IgG, IgG1and IgG3 frequencies to circumsporozoite protein (CSP) were similar across the age groups. IgG1 and IgG3 frequencies to thrombospondin-related adhesive protein (TRAP) were similar in all age groups as compared with total IgG that was higher in age group 04 months and 37-50 months (i = 8.93, P=0.0302). Total IgG and subclass responses to LSA-l showed a significant strong trend of increase across the age groups (X2 = 21.69, p<O.OOOI). On the other hand, frequencies of total IgG to apical membrane antigen-l (AMA-l), glycosylated and non-glycosylated erythrocyte binding antigens-175 (EBA 175 G and EBA-175 NG) were significantly higher in 0-4 months, decreased in 5-20 months and increased progressively up to 50 months (X: = 21.90,p=0.0001; X2 = 25.12, p<O.OOOIand X2 = 12.45,p=0.006 respectively). Only frequencies of IgGl to EBA-175 G was significantly different across the age groups (i = 6.24, p=0.0256), while IgG3 frequencies to these erythrocytic antigens were similar across the age groups with over 50% responding. MSP-119 results showed that total IgG frequencies to all MSP-l alleles were similar across the age group. Frequencies oflgGI to Q-KNG (X2 = 12.19,p=0.0256) and IgG3 to E-KNG, Q-TSR and E-TSR allelic peptides (X2 = 9.54, p=0.023; X2 = 9.59, p=0.0224, and X2 = 17.14, p=0.0007 respectively) significantly varied across age groups. The frequencies and levels of IgM responses to both pre-erythrocytic and erythrocytic XVI --------- antigens significantly increased across age groups (p<O.05) except for two MSP-l allelic peptide (Q-KNG and Q-TSR) that were similar across the age groups. This study suggests that age and lor exposure to malaria infection affects development of anti-malarial cytokines and antibodies and that there is differential acquisition of antibodies to pre-erythrocytic and erythrocytic antigens by children less than four years of age. These findings may be useful for future studies in malaria vaccine design and assessment and evolution of immunity in young children from malaria endemic areas.