Functional Analysis Reveals Geographical Variation in Inhibitory Immune Responses Against a Polymorphic Malaria Antigen

Abstract

Background: Plasmodium falciparum reticulocyte-binding protein homologue 2b (PfRh2b) is an invasion ligand that is a potential blood-stage vaccine candidate antigen; however, a naturally occurring deletion within an immunogenic domain is present at high frequencies in Africa and has been associated with alternative invasion pathway usage. Standardized tools that provide antigenic specificity in in vitro assays are needed to functionally assess the neutralizing potential of humoral responses against malaria vaccine candidate antigens.

Methods: Transgenic parasite lines were generated to express the PfRh2b deletion. Total immunoglobulin G (IgG) from individuals residing in malaria-endemic regions in Tanzania, Senegal, and Mali were used in growth inhibition assays with transgenic parasite lines.

Results: While the PfRh2b deletion transgenic line showed no change in invasion pathway utilization compared to the wild-type in the absence of specific antibodies, it outgrew wild-type controls in competitive growth experiments. Inhibition differences with total IgG were observed in the different endemic sites, ranging from allele-specific inhibition to allele-independent inhibitory immune responses.

Conclusions: The PfRh2b deletion may allow the parasite to escape neutralizing antibody responses in some regions. This difference in geographical inhibition was revealed using transgenic methodologies, which provide valuable tools for functionally assessing neutralizing antibodies against vaccine-candidate antigens in regions with varying malaria endemicity.

Keywords: PfRh2b; growth inhibition; invasion; transgenic; vaccine.

Figure 1.

The PfRh2b deletion does not affect alternative invasion pathway utilization but shows a competitive growth advantage over time. A, Quantitative PCR was employed to determine differences in invasion gene expression between DEL clones and WT control. CT values were compared relative to AMA-1 (∆CT). Data are expressed as fold change (∆∆CT) values relative to 3D7. Clag 3.2 is not shown as it is not expressed in the 3D7 clone used. B, Western blots of synchronous schizont material and invasion supernatants were performed. Samples were adjusted relative to the schizont stage–specific proteins PfAMA-1 for both schizont blots and supernatant blots. In schizonts, expected size of PfRh2b full-length protein (3D7, WT) is 382 kDa, which after proteolytic processing results in a 297 kDa cleavage product [45]; whereas the Deletion (DEL) results in a shorter protein which runs approximately 360 kDa with the cleavage product at 275 kDa. For PfRh2a, the unprocessed form runs at 370 kDa with a 285-kDa processed form. In supernatants, further processing results in an additional processed form of 290 kDa for PfRh2b (3D7, WT), 268 kDa (DEL), and 279 kDa for PfRh2a [45]. C, Invasion assays were performed to determine the erythrocyte receptor utilization for DEL clones compared to wild-type 3D7 and KO (3D7ΔPfRh2b). Percent invasion into enzyme-treated erythrocytes was calculated relative to invasion of the same parasite line into RPMI-treated erythrocytes. Assays were repeated 5 times, in triplicate, except for KO and DEL B (which were repeated twice, in triplicate). Values shown are the mean of all experiments; error bars represent SD. Overall significance is assessed by 1-way ANOVA; differences between columns are assessed with Dunnett test for multiple comparisons. Asterisks indicate significant differences (*P < .05; **P < .01; ***P < .001; ****P < .0001). KO invasion into lowT/Nm was tested, but is undetectable. D, Detailed measurements of invasion and growth were performed for PfRh2b deletion and control strains for 3 independent experiments, plated in duplicate. Erythrocytic cycle duration was measured for the 2 PfRh2b deletion (DEL A and DEL B) and control strains (3D7, WT) over 1 cycle. Stages were quantified morphologically at T = 0, T = 40, T = 48, and T = 56 hours postinvasion. A total of 300 parasitized cells were counted per time point; the average of the 3 experiments, plated in duplicate, is shown. E, Average merozoite number per schizont was calculated for transgenic parasites and controls. Data shown are from 3 independent experiments. Means and 95% CIs are shown. Overall significance is assessed by 1-way ANOVA; differences between columns are assessed with Dunnett test for multiple comparisons. Asterisks indicate significant differences (*P < .05; **P < .01; ***P < .001). F, Competitive growth assays were conducted in which DEL and WT were mixed in equal proportions and kept in in vitro culture for 1 month, with weekly gDNA harvests. Relative proportions of DEL and WT were assessed by Southern blot hybridization using a probe to human DHFR. A representative experiment is shown with DEL B and WT. Relative ratios of WT compared to DEL over time were calculated for both DEL A and DEL B clones, with 2 independent experiments per clone; error bars represent the range of the 2 experiments. Abbreviations: ANOVA, analysis of variance; CIs, confidence intervals; CT, cycle threshold; DEL, PfRh2b deletion single crossover parasites; DHFR, dihydrofolate reductase; gDNA, genomic DNA; kDa, kilodalton; KO, 3D7∆2b, C2 parasites; lowT, low trypsin-treated erythrocytes; Nm, neuraminidase-treated erythrocytes; PCR, polymerase chain reaction; PfRh2b, Plasmodium falciparum reticulocyte-binding protein homologue 2b; RPMI, Roswell Park Memorial Institute; T, trypsin-treated erythrocytes; WT, PfRh2b wild-type replacement parasite line (1A).

Figure 2.

PfRh2b deletion reveals region-specific differences in immune-mediated inhibition of invasion. A, Purified IgG from Tanzania, Senegal, and Mali were used in highly standardized GIAs with transgenic parasite lines (WT, DEL A, and KO). Inhibition means and 95% CIs are shown. B–D, Inhibitory profiles in each endemic region show differences by strain. Inhibition is displayed as “Inhibition ∆” showing the difference between strains. On the left of each graph is the Inhibition ∆ for WT-KO, with any differences representing differences in inhibition of the PfRh2b (WT) and non-PfRh2b (KO) invasion pathways; in the middle, the Inhibition ∆ are shown for specific allelic inhibition of either PfRh2b full-length (WT) compared to PfRh2b deletion (DEL); and on the right, Inhibition ∆ are shown for specific allelic inhibition of either PfRh2b deletion (DEL) compared to KO. Significant P values from comparing inhibition of WT to DEL A, and DEL A to KO, using nonparametric-paired t tests (Wilcoxon matched pairs) are indicated. Abbreviations: CIs, confidence intervals; DEL, PfRh2b deletion single crossover parasites; GIAs, growth inhibition assays; IgG, immunoglobulin G; KO, 3D7∆2b, C2 parasites; ns, not significant; PfRh2b, Plasmodium falciparum reticulocyte-binding protein homologue 2b; WT, PfRh2b wild-type replacement parasite line (1A).

Figure 3.

Model of PfRh2b-dependent and -independent inhibitory immune responses. Examples of immune neutralization that is either PfRh2b allele-specific or allele transcendent is illustrated using individual samples from Senegal (A) and all samples from Tanzania, Senegal, and Mali (B). A significant difference is considered greater than 15% difference in inhibition, the degree of difference tolerated in replicates of the same strain in the GIA. A, Situation A shows no significant difference in immune inhibition between the 3 transgenic strains, whether low inhibition or high inhibition, termed allele-independent immunity. Situations B–F demonstrate differences in inhibition specifically due to the allelic form of PfRh2b (allele-specific immunity). Situation B shows increased inhibition of DEL relative to the WT and KO. Such a result could indicate that the deletion in the protein is revealing an alternative epitope of PfRh2b. In situation C, there is an “immune escape” phenotype observed for both the PfRh2b deletion as well as the KO, implying that the differences in inhibition observed are specifically due to the allele of PfRh2b present. Situation D shows “immune escape” for the PfRh2b KO only. Situation E shows enhanced inhibition of both the WT and the deletion allele, and situation F shows incremental increases in inhibition from WT, to PfRh2b deletion, to PfRh2b KO. B, The fraction of the total for each type of inhibitory situation is shown for each population. The population genetic prevalence of the PfRh2b full-length and PfRh2b deletion alleles are shown below as bars. Abbreviations: DEL, PfRh2b deletion single crossover parasites; GIA, growth inhibition assay; KO, 3D7∆2b, C2 parasites; PfRh2b, Plasmodium falciparum reticulocyte-binding protein homologue 2b; WT, PfRh2b wild-type replacement parasite line (1A).