The Antibody Response of Pregnant Cameroonian Women to VAR2CSA ID1-ID2a, a Small Recombinant Protein Containing the CSA-Binding Site

<div><p>In pregnant women, <i>Plasmodium falciparum</i>-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women naturally acquire antibodies to ID1-ID2a and if antibodies to ID1-ID2a correlate with absence of placental malaria at delivery. Antibody levels to full-length VAR2CSA and ID1-ID2a were measured in plasma samples from 745 pregnant Cameroonian women, 144 Cameroonian men, and 66 US subjects. IgM levels and IgG avidity to ID1-ID2a were also determined. As expected, antibodies to ID1-ID2a were absent in US controls. Although pregnant Cameroonian women developed increasing levels of antibodies to full-length VAR2CSA during pregnancy, no increase in either IgM or IgG to ID1-ID2a was observed. Surprisingly, no differences in antibody levels to ID1-ID2a were detected between Cameroonian men and pregnant women. For example, in rural settings only 8–9% of males had antibodies to full-length VAR2CSA, but 90–96% had antibodies to ID1-ID2a. In addition, no significant difference in the avidity of IgG to ID1-ID2a was found between pregnant women and Cameroonian men, and no correlation between antibody levels at delivery and absence of placental malaria was found. Thus, the response to ID1-ID2a was not pregnancy specific, but predominantly against cross-reactivity epitopes, which may have been induced by other PfEMP1 antigens, malarial antigens, or microbes. Currently, ID1-ID2a is a leading vaccine candidate, since it binds to the CSA with the same affinity as the full-length molecule and elicits binding-inhibitory antibodies in animals. Further studies are needed to determine if the presence of naturally acquired cross-reactive antibodies in women living in malaria endemic countries will alter the response to ID1-ID2a following vaccination with ID1-ID2a.</p></div

Comparison of Ab levels between FV2 and ID1-ID2a.

<p><b>A–C</b>) Ab levels to FV2, DBL3, DBL5 and ID1-ID2a FCR3 were measured in plasma of 116 PM+ and 348 PM- pregnant women living in city of Yaoundé. In addition, comparisons were made in Ab levels to FV2 and ID1-ID2a in <b>D</b>) males in Yaoundé, <b>E</b>) males in the village of Ngali II, and <b>F</b>) males in Simbok village. Positive correlations were found for all comparisons; Pearson's correlation coefficients (r) are reported.</p

IgG levels to ID1-ID2a in Cameroonian women during pregnancy.

<p>Ab levels to FV2 (<b>A, D</b>), ID1-ID2a 3D7 (<b>B, E</b>), and ID1-ID2a FCR3 (<b>C, F</b>) were measured in plasma of pregnant women living in the city of Yaoundé (<b>A-C</b>) and village of Ngali II (<b>D–F</b>). Samples were randomly selected within a trimester so only one data point per women per trimester was included. Number of samples per trimester ranged from: <b>A–C</b>: Primigravidae (PG) n = 33–39, multigravidae (MG) n = 63–74; <b>D–F</b>: PG n = 13–15, MG n = 33–68. To determine if Ab levels increased during pregnancy, data for women with samples collected in the first, second, and third trimesters (City: PG n = 32, MG n = 53; Village: PG n = 7, MG n = 17) were assessed using the Friedman's test. As expected, Ab levels to FV2 increased during pregnancy in the village (Fig. <b>1B</b>: PG: p = 0.008, MG p = 0.001). Although a similar trend was observed in the city, the increase was not significant, probably due to low transmission (<b>Fig. 1A</b>). Likewise, higher Ab levels to FV2 were observed in PG and MG compared to males in the city (all p values <0.001) and village (all p values <0.0001). In contrast, Ab levels to ID1-ID2a (both strains) did not increase during pregnancy in either location (p>0.05) and no significant differences were found in Ab levels to ID1-ID2a (3D7 or FCR3) between males and females in the two sites (all p values>0.05) (Fig. 1B–1F). Although statistically significant higher Ab levels to ID1-ID2a 3D7 were found in MG in the village compared to males (Fig. <b>E</b>, p<0.05), the trend was not found with the FCR3 strain. ♂- males, small ♀- PG, big ♀- MG; I, II, III trim  =  first, second and third trimester, respectively. Median MFI and Inter-Quartile Range (IQR) are plotted.</p

IgM levels to FV2 and ID1-ID2a in Cameroonian women.

<p>IgM levels to FV2 and ID1-ID2a (3D7 and FCR3) were measured in plasma collected at first smear-positive visit and the following visit approximately one month later from women living in <b>A</b>) city of Yaoundé (PG n = 17, MG n = 26) and <b>B</b>) village of Ngali II (PG n = 21, MG n = 33). No statistically significant differences in IgM levels to either ID1-ID2a or FV2 were observed between PG and MG in both locations (all p values>0.05). Since the beads were coupled at saturation, it appears that both PG and MG produced higher IgM levels to FV2 compared to ID1-ID2a 3D7 (both locations p-values <0.001). Yaoundé PG and MG produced higher IgM levels to FV2 compared to ID1-ID2a FCR3 (p<0.05). Small ♀- PG, big ♀- MG. Median MFI and IQR are plotted.</p

Prevalence of IgG to FV2 and ID1-ID2a in non-pregnant individuals and pregnant women at delivery.

a<p>Cut-off for FV2 seropositivity was determined based on the mean + 2 SD for resident males after excluding the occasional male (n = 2 Yaoundé, n = 1 Ngali, n = 1 Simbok) with MFI greater than the mean +3 SD.</p>b<p>US controls (n = 66) were used to establish a cut-off for seropositivity to ID1-ID2a.</p>c<p>3% of women who were not seropositive for FV2 were seropositive for DBL5 FCR3, indicating they had been exposed to CSA-binding <i>P. falciparum</i> IE.</p>d<p>Mean ± SD.</p

IgG levels to N-terminal domains of FV2 and DBL5 in the city of Yaoundé.

<p>Ab levels to DBL1 (3D7 and 7G8), DBL1+2 (7G8), DBL2 FCR3 and DBL5 FCR3 in the plasma of 116 PM+ and 348 PM− multigravidae women living in the city of Yaoundé were measured at delivery (cross-sectional cohort). Both PM+ and PM− women had significantly higher levels of Ab to DBL5 compared to Ab to DBL1, DBL 1+2 and DBL 2 (all p values <0.0001). PM+ and PM− multigravidae had significantly higher levels of Ab to DBL1 3D7, DBL1 7G8, DBL1+2 and DBL5 compared to males (all p values <0.0001). Only a marginally significant differences was found in Ab levels to DBL2 between males and females (p = 0.051). No statistical differences were found in Ab levels to any of the domains between PM+ and PM− women (all p values>0.05). Open circles represent PM− and black circles represent PM+. Median and IQR are also plotted.</p

Proportion of high avidity IgG to FV2 and ID1-ID2a.

<p>A subset of women in Yaoundé and Ngali II with Ab ID1-ID2a were tested in the avidity assay. The proportion of Ab that remained bound to <b>A</b>) FV2, <b>B</b>) ID1-ID2a 3D7, and <b>C</b>) ID1-ID2a FCR3 in the presence of chaotropic salt was measured in samples collected in city of Yaoundé (n = 6 males, n = 18 women) and village of Ngali II (n = 9 males, n = 20 women). Although the mean proportion of high avidity Ab to FV2 was significantly higher in Ngali II pregnant women compared to males (*** p = 0.0003), no statistically significant differences were found in Ab avidity to ID1-ID2a (3D7 and FCR3) between males and females, both in the city and village (all p>values 0.05). ♂- males, ♀- females Median and IQR are also plotted.</p

IgG levels to ID1-ID2a in different cohorts.

<p>IgG levels to FV2 and ID1-ID2a (3D7 and FCR3) were measured in plasma collected at delivery from women living in <b>A</b>) city of Yaoundé (n = 35 males, n = 33 PG, n = 63 MG), <b>B</b>) village of Ngali II (n = 58 males, n = 15 PG, n = 68 MG), <b>C</b>) Simbok village (n = 51 males, n = 102 women, and <b>D</b>) in a cross-sectional cohort of MG in Yaoundé (PM+n = 116, PM− n = 348), as well as, US pregnant (n = 42) and non-pregnant subjects (n = 24). Major findings included: 1) significantly higher Ab levels to ID1-ID2a (both strains) in Cameroonian males (city and village) compared to US pregnant women (p<0.001); 2) similar IgG levels to FV2 in US pregnant and non-pregnant controls and males in Yaoundé; but, higher levels in Cameroonian males living in the village (p<0.0001), suggesting repeated infection induced Ab to FV2 in some males; 3) no significant differences in IgG to ID1-ID2a in Cameroonian males and females living in the city and village (both strains p> values 0.05); and 4) no significant differences in IgG to ID1-ID2a FCR3 between PM+ and PM- MG in the city (<b>D</b>: p = 0.46). Horizontal bars represent median, first and third quartiles, and vertical bars represent Inter-Quartile Range (IQR). □- US non-pregnant (US NP), ▪ US pregnant women (US PW), ♂- Cameroonian males (M), small ♀- Cameroonian PG, big ♀- Cameroonian MG. Median MFI and IQR are plotted.</p

Colorimetric Detection of <i>Plasmodium vivax</i> in Urine Using MSP10 Oligonucleotides and Gold Nanoparticles

<div><p><i>Plasmodium vivax</i> is the most prevalent cause of human malaria in the world and can lead to severe disease with high potential for relapse. Its genetic and geographic diversities make it challenging to control. <i>P</i>. <i>vivax</i> is understudied and to achieve control of malaria in endemic areas, a rapid, accurate, and simple diagnostic tool is necessary. In this pilot study, we found that a colorimetric system using AuNPs and MSP10 DNA detection in urine can provide fast, easy, and inexpensive identification of <i>P</i>. <i>vivax</i>. The test exhibited promising sensitivity (84%), high specificity (97%), and only mild cross-reactivity with <i>P</i>. <i>falciparum</i> (21%). It is simple to use, with a visible color change that negates the need for a spectrometer, making it suitable for use in austere conditions. Using urine eliminates the need for finger-prick, increasing both the safety profile and patient acceptance of this model.</p></div

MSP10 N-Terminal oligonucleotide and AuNPs were able to detect <i>P</i>. <i>vivax</i> in urine with higher sensitivity, specificity and lower cross-reactivity than C-Terminal oligonucleotide of MSP10.

<p>MSP10 N-Terminal oligonucleotide and AuNPs were able to detect <i>P</i>. <i>vivax</i> in urine with higher sensitivity, specificity and lower cross-reactivity than C-Terminal oligonucleotide of MSP10.</p