The Dantu blood group prevents parasite growth in vivo: Evidence from a controlled human malaria infection study
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eLife assessment
The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria since it increases the surface tension of red blood cells making it harder for malaria parasites to invade. In this important work, the authors show that parasite growth is indeed restricted in vivo by testing this hypothesis in adult Kenyan volunteers infected with P. falciparium under careful monitoring. They were able to show convincingly that indeed, parasite growth was reduced amongst Dantu adults.
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Abstract
The long co-evolution of Homo sapiens and Plasmodium falciparum has resulted in the selection of numerous human genetic variants that confer an advantage against severe malaria and death. One such variant is the Dantu blood group antigen, which is associated with 74% protection against severe and complicated P. falciparum malaria infections in homozygous individuals, similar to that provided by the sickle haemoglobin allele (HbS). Recent in vitro studies suggest that Dantu exerts this protection by increasing the surface tension of red blood cells, thereby impeding the ability of P. falciparum merozoites to invade them and reducing parasite multiplication. However, no studies have yet explored this hypothesis in vivo .
Methods:
We investigated the effect of Dantu on early phase P. falciparum (Pf) infections in a controlled human malaria infection (CHMI) study. 141 sickle-negative Kenyan adults were inoculated with 3.2 × 10 3 aseptic, purified, cryopreserved Pf sporozoites (PfSPZ Challenge) then monitored for blood-stage parasitaemia for 21 days by quantitative polymerase chain reaction (qPCR)analysis of the 18S ribosomal RNA P. falciparum gene. The primary endpoint was blood-stage P. falciparum parasitaemia of ≥500/μl while the secondary endpoint was the receipt of antimalarial treatment in the presence of parasitaemia of any density. On study completion, all participants were genotyped both for Dantu and for four other polymorphisms that are associated with protection against severe falciparum malaria: α + -thalassaemia, blood group O, G6PD deficiency, and the rs4951074 allele in the red cell calcium transporter ATP2B4 .
Results:
The primary endpoint was reached in 25/111 (22.5%) non-Dantu subjects in comparison to 0/27 (0%) Dantu heterozygotes and 0/3 (0.0%) Dantu homozygotes (p=0.01). Similarly, 49/111 (44.1%) non-Dantu subjects reached the secondary endpoint in comparison to only 7/27 (25.9%) and 0/3 (0.0%) Dantu heterozygotes and homozygotes, respectively (p=0.021). No significant impacts on either outcome were seen for any of the other genetic variants under study.
Conclusions:
This study reveals, for the first time, that the Dantu blood group is associated with high-level protection against early, non-clinical, P. falciparum malaria infections in vivo . Learning more about the mechanisms involved could potentially lead to new approaches to the prevention or treatment of the disease. Our study illustrates the power of CHMI with PfSPZ Challenge for directly testing the protective impact of genotypes previously identified using other methods.
Funding:
The Kenya CHMI study was supported by an award from Wellcome (grant number 107499). SK was supported by a Training Fellowship (216444/Z/19/Z), TNW by a Senior Research Fellowship (202800/Z/16/Z), JCR by an Investigator Award (220266/Z/20/Z), and core support to the KEMRI-Wellcome Trust Research Programme in Kilifi, Kenya (203077), all from Wellcome. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.
Clinical trial number:
NCT02739763
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Author Response
Reviewer #1 (Public Review):
The human genetic variant Dantu increases the surface tension of red blood cells making it hard for malaria parasites to invade. This was shown beautifully by Kariuki et al in 2020 (doi.org/10.1038/s41586-020-2726-6) by analysing blood from children using in vitro assays with cultured malaria parasites. Now Kariuki et al show that parasite growth is indeed restricted in vivo by infecting Dantu adults under controlled conditions with cryopreserved Plasmodium falciparum sporozoites and analysing parasite growth by qPCR. The authors compare parasite growth, peak parasitaemia and if / when treatment was sought for malaria symptoms between non-Dantu (111) and Dantu heterozygous (27) and homozygous (3) participants. Dantu either completely prevented malaria parasite detection in the blood (for …
Author Response
Reviewer #1 (Public Review):
The human genetic variant Dantu increases the surface tension of red blood cells making it hard for malaria parasites to invade. This was shown beautifully by Kariuki et al in 2020 (doi.org/10.1038/s41586-020-2726-6) by analysing blood from children using in vitro assays with cultured malaria parasites. Now Kariuki et al show that parasite growth is indeed restricted in vivo by infecting Dantu adults under controlled conditions with cryopreserved Plasmodium falciparum sporozoites and analysing parasite growth by qPCR. The authors compare parasite growth, peak parasitaemia and if / when treatment was sought for malaria symptoms between non-Dantu (111) and Dantu heterozygous (27) and homozygous (3) participants. Dantu either completely prevented malaria parasite detection in the blood (for 21 days) or slowed down parasite growth considerably.
The authors present compelling in vivo evidence that Dantu conveys protection by preventing malaria parasites from establishing a blood-stage infection. Because the effect on parasite growth is crystal clear the link to uncomplicated malaria follows - no/less parasites leads to less participants experiencing malaria symptoms and seeking treatment. It should however be noted that the paper does not show that Dantu reduces symptomatology at identical parasite densities to non-Dantu. Its protective effect seems to be purely parasitological.
Given that all volunteers were exposed to malaria prior to being experimentally infected (in various transmission settings ranging from low to high) the authors state that they adjusted for factors like schizont antibody concentration in their multi-variate analysis. More details on the assumptions and which dependent / independent variables were included would benefit interpretation. It would be also good to see if Dantu individuals were spread homogeneously across all transmission settings - if e.g. they all had history of intense malaria exposure and thus strong pre-existing anti-malaria immunity this might account in part for reduced parasite growth when compared to non-Dantu from lower transmission settings. Being able to de-convolute the effect of pre-existing immunity from Dantu would strengthen the paper.
Thank you for the positive feedback and summary of the key findings. We absolutely agree that breaking down the impact of Dantu genotype by transmission would have been very interesting, but the sample numbers for some of the genotypic groups were simply too small to make stratification by area of residence meaningful. Instead, to address the core issue of whether prior immunity is a complicating factor in our analysis, we used measurements of antibodies to whole schizont extract as a proxy indicator of transmission setting or “malaria exposure” in our multivariate analyses. There was no difference in anti-schizont antibody levels across Dantu genotype groups – these data are now included in Figure 3 – figure supplement 1, as requested. This suggests that differences in pre-existing anti-malaria immunity between Dantu and non-Dantu cannot explain the differences seen in our current study. Regarding the comment about assumptions and variables in the multivariate analysis, we have added more details as requested, as outlined in further detail in subsequent points below.
The authors also presents data on other red cell polymorphisms known to modulate malaria infection and improve outcome: G6PD, blood group O, alpha thalassaemia and ATP2B4. However, no statistically significant differences between non-carriers and hetero/homozygous individuals were observed. This is probably because these mutations exert their effect not directly on parasite growth but modulate disease symptoms when parasite burden is high - which cannot be investigated in controlled human malaria infection settings as ethical considerations mandate treatment of all volunteers at parasite densities >500 parasites/ ul or any parasitaemia with symptoms. Controlled infections need to be complemented with other methods to understand the protective impact of genetic polymorphisms.
We thank the reviewer for this helpful observation with which we completely agree. To acknowledge this issue, we have added some consideration of this point to the Discussion section of the revised manuscript, within the sub-section that discusses protective mechanisms of other red cell polymorphisms on page 14.
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eLife assessment
The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria since it increases the surface tension of red blood cells making it harder for malaria parasites to invade. In this important work, the authors show that parasite growth is indeed restricted in vivo by testing this hypothesis in adult Kenyan volunteers infected with P. falciparium under careful monitoring. They were able to show convincingly that indeed, parasite growth was reduced amongst Dantu adults.
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Reviewer #1 (Public Review):
The human genetic variant Dantu increases the surface tension of red blood cells making it hard for malaria parasites to invade. This was shown beautifully by Kariuki et al in 2020 (doi.org/10.1038/s41586-020-2726-6) by analysing blood from children using in vitro assays with cultured malaria parasites. Now Kariuki et al show that parasite growth is indeed restricted in vivo by infecting Dantu adults under controlled conditions with cryopreserved Plasmodium falciparum sporozoites and analysing parasite growth by qPCR. The authors compare parasite growth, peak parasitaemia and if / when treatment was sought for malaria symptoms between non-Dantu (111) and Dantu heterozygous (27) and homozygous (3) participants. Dantu either completely prevented malaria parasite detection in the blood (for 21 days) or slowed …
Reviewer #1 (Public Review):
The human genetic variant Dantu increases the surface tension of red blood cells making it hard for malaria parasites to invade. This was shown beautifully by Kariuki et al in 2020 (doi.org/10.1038/s41586-020-2726-6) by analysing blood from children using in vitro assays with cultured malaria parasites. Now Kariuki et al show that parasite growth is indeed restricted in vivo by infecting Dantu adults under controlled conditions with cryopreserved Plasmodium falciparum sporozoites and analysing parasite growth by qPCR. The authors compare parasite growth, peak parasitaemia and if / when treatment was sought for malaria symptoms between non-Dantu (111) and Dantu heterozygous (27) and homozygous (3) participants. Dantu either completely prevented malaria parasite detection in the blood (for 21 days) or slowed down parasite growth considerably.
The authors present compelling in vivo evidence that Dantu conveys protection by preventing malaria parasites from establishing a blood-stage infection. Because the effect on parasite growth is crystal clear the link to uncomplicated malaria follows - no/less parasites leads to less participants experiencing malaria symptoms and seeking treatment. It should however be noted that the paper does not show that Dantu reduces symptomatology at identical parasite densities to non-Dantu. Its protective effect seems to be purely parasitological.
Given that all volunteers were exposed to malaria prior to being experimentally infected (in various transmission settings ranging from low to high) the authors state that they adjusted for factors like schizont antibody concentration in their multi-variate analysis. More details on the assumptions and which dependent / independent variables were included would benefit interpretation. It would be also good to see if Dantu individuals were spread homogeneously across all transmission settings - if e.g. they all had history of intense malaria exposure and thus strong pre-existing anti-malaria immunity this might account in part for reduced parasite growth when compared to non-Dantu from lower transmission settings. Being able to de-convolute the effect of pre-existing immunity from Dantu would strengthen the paper.
The authors also presents data on other red cell polymorphisms known to modulate malaria infection and improve outcome: G6PD, blood group O, alpha thalassaemia and ATP2B4. However, no statistically significant differences between non-carriers and hetero/homozygous individuals were observed. This is probably because these mutations exert their effect not directly on parasite growth but modulate disease symptoms when parasite burden is high - which cannot be investigated in controlled human malaria infection settings as ethical considerations mandate treatment of all volunteers at parasite densities >500 parasites/ ul or any parasitaemia with symptoms. Controlled infections need to be complemented with other methods to understand the protective impact of genetic polymorphisms.
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Reviewer #2 (Public Review):
The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria. The proposed mechanism of protection is reduced red cell invasion resulting in reduced parasite multiplication. This hypothesis was tested in adult Kenyan volunteers infected with P. falciparum under careful monitoring. The strength of the study is that the CHMI model using a single parasite strain has few confounders and it provides a very clear answer. The data reported on the other "protective" genetic polymorphisms is also fascinating. The hypothesis that Dantu reduces merozoite invasion has some support from previous laboratory studies, but it would be useful to confirm, once invasion is successful, that intraerythrocytic growth is unimpaired (e.g. …
Reviewer #2 (Public Review):
The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria. The proposed mechanism of protection is reduced red cell invasion resulting in reduced parasite multiplication. This hypothesis was tested in adult Kenyan volunteers infected with P. falciparum under careful monitoring. The strength of the study is that the CHMI model using a single parasite strain has few confounders and it provides a very clear answer. The data reported on the other "protective" genetic polymorphisms is also fascinating. The hypothesis that Dantu reduces merozoite invasion has some support from previous laboratory studies, but it would be useful to confirm, once invasion is successful, that intraerythrocytic growth is unimpaired (e.g. count merozoites per schizont, measure asexual cycle length etc).
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