The Quantity and Quality of African Children's IgG Responses to Merozoite Surface Antigens Reflect Protection against Plasmodium falciparum Malaria

Contains fulltext : 81196.pdf (publisher's version ) (Open Access)BACKGROUND: Antibodies, particularly cytophilic IgG subclasses, with specificity for asexual blood stage antigens of Plasmodium falciparum, are thought to play an important role in acquired immunity to malaria. Evaluating such responses in longitudinal sero-epidemiological field studies, allied to increasing knowledge of the immunological mechanisms associated with anti-malarial protection, will help in the development of malaria vaccines. METHODS AND FINDINGS: We conducted a 1-year follow-up study of 305 Senegalese children and identified those resistant or susceptible to malaria. In retrospective analyses we then compared post-follow-up IgG responses to six asexual-stage candidate malaria vaccine antigens in groups of individuals with clearly defined clinical and parasitological histories of infection with P. falciparum. In age-adjusted analyses, children resistant to malaria as well as to high-density parasitemia, had significantly higher IgG1 responses to GLURP and IgG3 responses to MSP2 than their susceptible counterparts. Among those resistant to malaria, high anti-MSP1 IgG1 levels were associated with protection against high-density parasitemia. To assess functional attributes, we used an in vitro parasite growth inhibition assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1, MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked age-related decline was observed in these effects. CONCLUSION: Our data are consistent with the idea that protection against P. falciparum malaria in children depends on acquisition of a constellation of appropriate, functionally active IgG subclass responses directed to multiple asexual stage antigens. Our results suggest at least two distinct mechanisms via which antibodies may exert protective effects. Although declining with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The latter could act on parasite growth via indirect parasiticidal pathways

IgG antibody levels in all participants.

<p>Total and cytophilic IgG subclass (IgG1 & IgG3) levels of 273 individuals (<i>HBB</i> AS carriers excluded) with specificity for the panel of asexual blood stage antigens of <i>P. falciparum</i>. Box-whisker plots represent medians with 25^th and 75^th percentiles (boxes), and with 10^th and 90^th percentiles (whiskers), outliers as discrete dots. The prevalence of positive responders to each recombinant proteins is noticed between brackets. A positive responder was defined as an individual who had a level of specific IgG over the positivity thresholds. These positivity thresholds were determined, for total IgG, IgG1 and IgG3 to each recombinant proteins, from the mean reactivities+2 SD of 30 plasma samples from Dutch non-immune volunteers.</p

IgG antibody isotype responses in groups of children with and without malaria attacks.

<p>IgG1 (A) and IgG3 (B) responses to a panel of recombinant proteins corresponding to 5 different <i>P. falciparum</i> asexual stage antigens in groups of children segregated according to their status as either low (ACLP, <2500 parasites/ul) or high (ACHP, ≥2500 parasites/ul) asymptomatic parasitemia carriers, or those with one or more malaria attacks (UMA, parasitemia plus fever) during 12 months' follow-up. Box-whisker plots represent medians with 25^th and 75^th percentiles (boxes), and with 10^th and 90^th percentiles (whiskers), outliers as discrete dots.</p

Associations between IgG responses and anti-disease/anti-parasite immunity.

*<p>The effect of malariometric status on IgG responses (IgGt: total IgG), determined by ANCOVA, was adjusted for age.</p>**<p>Significantly different after adjustment for multiple tests (Bonferroni correction, threshold of significance: P = 0.002).</p>***<p>Logistic regression was applied for the analysis of associations with protection against malaria attacks, where status (UMA vs ACLP groups) was used as the dependent variable against the explanatory variables (IgG subtype responses (IgG3 to MSP2 FC27 and IgG1 to GLURP) and age) shown to be significantly associated by ANCOVA. OR values were assessed for 10 AU increased. Age was also associated with malaria protection in the analysis (p = 0.011, OR = 0.84).</p

Parasite growth inhibitory capacity of purified IgG in groups segregated according to malariometric status.

<p>Data from IgG purified from a total of 75 plasma samples is illustrated for groups of children segregated according to their status as either low (ACLP, <2500 parasites/ul) or high (ACHP, ≥2500 parasites/ul) asymptomatic parasitemia carriers, or those with one or more malaria attacks (UMA, parasitemia plus fever) during 12 months' follow-up. 15 samples (5 matched trios) were excluded due to large variations between triplicate samples (coefficient of variation >30%). Box-whisker plots represent medians with 25^th and 75^th percentiles (boxes), and with 10^th and 90^th percentiles (whiskers), outliers as discrete dots. Doted lines represent the arithmetic mean of parasite growth inhibition.</p

The association between parasite growth inhibitory activity of purified IgG and age.

<p>Parasite growth inhibitory (PGI) capacity of purified IgG plotted against age, with regression line fitted (using the regression fit command of STATA software). A significant difference in PGI (p = 0.03, Kruskall-Wallis test) was observed between groups segregated according to age (8–9 years vs 10–11 years vs 12–14 years).</p

Associations between IgG responses and protection against malaria attacks.

*<p>Presented are only those antibody responses, from the 20 tested, found to be significantly associated with malariometric status (P<0.05 before multiple test correction); IgGt: total IgG.</p>**<p>The effect of malariometric status on IgG responses, determined by ANCOVA, was adjusted for age.</p>***<p>Significantly different after adjustment for multiple tests (Bonferroni correction, threshold of significance: P = 0.002).</p>†<p>Logistic regression was used to assess associations with protection against malaria attacks, where status (UMA vs AC groups) was used as the dependent variable against the explanatory variables (IgG subtype responses/age) shown to be significantly associated by ANCOVA. OR values were assessed for 10 AU increased.</p><p>Note: age was also independently associated with malaria protection in this analysis (P = 0.034, OR = 0.89).</p

Comparison of IgG levels in groups of samples with high or low parasite growth inhibition (PGI).

*<p>No significant difference in age was observed between the groups of samples showing high and low level of PGI (p = 0.33, Mann-Whitney).</p>**<p>Non parametric Mann-Whitney test was used to compare IgG levels in the groups.</p>***<p>Significant p value after multiple test adjustment (Bonferroni correction).</p

Participants' characteristics.

<p>UMA = uncomplicated malaria group; AC = asymptomatic carrier group; ACHP = asymptomatic carrier subgroup with high parasitemia; ACLP = asymptomatic carrier subgroup with low parasitemia; RBC = red blood cells.</p>*<p>Significant age difference between UMA, AC and uninfected groups (Anova, p<0.0001).</p>**<p>No significant gender difference in the groups (Chi2, p = 0.513).</p>***<p>No significant difference between groups (Chi2, p = 0.25).</p>****<p>The Hbb AS genotype was significantly less frequent in the UMA group than the group of individuals free of clinical malaria (Chi2, p = 0.049).</p>†<p>Excludes the uninfected group (33 individuals).</p