The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen

Abstract

Background: Plasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (< 0.5 infections/person/year). Purportedly non-immune individuals with discrete (non-overlapping) P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this population's antibody response to PfMSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for PfMSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of PfMSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to PfMSP1-19KD are determined.

Methods: The 42KD region in PfMSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2) was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether PfMSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of PfMSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types.

Results: Despite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4%) were the Q-KSNG-F haplotype and 1 (0.6%) was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of PfMSP1-19KD.

Conclusions: A non-allele specific antibody response in PfMSP1-19KD may explain why other allelic forms have not been maintained or evolved in this population. This has important implications for the use of PfMSP1-19KD as a vaccine candidate. It is possible that Peruvians have increased antibody responses to the shared sites of PfMSP1-19KD, either due to exposure/parasite characteristics or due to a human-genetic predisposition. Alternatively, these allelic polymorphisms are not immune-specific even in other geographic regions, implying these polymorphisms may be less important in immune evasion that previous studies suggest.

Figure 1

A-C - Genotyping methods for Pfmsp1-42KD and Pfmsp1-B2. A) Schema of Pfmsp1, indicating contrasting regions of conservation and variability. B) Highlights the Pfmsp1-42KD region of Pfmsp1. The polymorphic nature of Pfmsp1-33KD is indicated by the darker shades at the C-terminal end of the Mad20 allele (tan) and K1 allele (blue), while the lighter shades are used to indicate increased conservation. Colored arrows, defined in the figure, are used to illustrate the position of the primers used in PCR amplification. The colored arrows, with the same definition as found in (A), are used to illustrate the combination of primers (Mad20 or K1) used when testing for permutations that may have been observed due to interfamily sexual recombination. C) Highlights the three allelic families detected as a result of using the Roberts et al [22] method. Stemming from the polymorphic Pfmsp1B2 region, illustrated as the red block, are those three allelic families: Mad20 (tan), K1 (blue), RO33 (pink). Darker shades at the center of Mad20 and K1 are used to indicate increased genetic variation due to it the presence of a variable repeat-length region. Colored arrows, defined in the figure, are used to illustrate the position of the primers used in PCR amplification. The colored arrows, with the same definition as found in (A), are used to illustrate the combination of primers (5' or 3') used when testing for permutations that may have been observed due to interfamily sexual recombination.

Figure 2

Experimental design for Antibody depletion ELISA. A representative sample is shown. Patients' sera were plated in duplicate and in 4 replicates in the first row on each type of "primary" plate (E-KNG, Q-KNG, E-TSR, and Q-TSR). The sera were then transferred down the plate seven times, incubating for half an hour before each transfer. After incubating the patient sera in the last row of the primary plate, it was transferred to a secondary plate of each allele.

Figure 3

Tabular comparison of concordance between the four Pf MSP1-19KD alleles. N = 105, dark grey = high positive antibody response (values greater than 2 * negative cut-off for each allele), medium grey = medium positive response (values between 1.5 * the negative cut-off and 2 * the negative cut-off), light grey = low positive response (values between the negative cut-off and 1.5 * negative cut-off), and white = negative antibody response (values less than the negative cut-off). Each row represents a different code/date.

Figure 4

Immunodepletion results. The mean residual antibody OD values are shown on the y-axis, antigens that underwent immunodepletion are shown on the z-axis, and secondary antibody responses are shown on the x-axis. The range for all samples was 0.019-0.142. The standard error of most comparisons was low (with a range of 0.00-0.05), and so not shown on this graphic.