Sunday, October 18, 2015

How Blood Group O Protects Against Malaria

It was the pioneer work by Oliver Gonzalez (1944) where it was postulated that plasmodia possess antigens equivalent to the blood-group antigens A and B of man. The work referred showed that the anti-A titre of Group O subjects, who had often had attacks of Plasmodium vivax and Plasmodium falciparum malaria, was far greater than in control Group O subjects; and also that the anti-B titre of the infected subjects was raised, though not to the same extent as for anti-A [1]. His theory was not totally accepted nor was it denied [2]. Since, that time till early 2000, the same dilemma persisted, whether to accept this theory or totally reject it.

Though it was well documented that people with blood type O are protected against severe malaria [3], while those with other types, such as A, often fall into a coma and die. Then there were reports that suggest otherwise, such as where blood group A, B and O were equally susceptible to malaria infection, AB blood group had less number of persons with malaria parasites. A significantly lower frequency of Plasmodium falciparum was observed among individuals with blood groups A and O. In other two blood groups B and AB, no difference in P. vivax and P. falciparum proportions were observed. A two-year study showed that the frequency of repeated attacks between all blood groups was similar [4].

Understanding the mechanisms behind this has been one of the main goals of malaria research. Although it has been known for two decades that human red cell ABO blood group affects the ability of malaria parasites to form rosettes [5], the precise details of the interaction remained obscure. Rosettes are clusters of infected red blood cells binding to uninfected red blood cells. 

Rosetting is characterized by the binding of P. falciparum-infected red blood cells (RBCs) to uninfected RBCs to form clusters of cells that are thought to contribute to the pathology of falciparum malaria by obstructing blood flow in small blood vessels.

Many previous work had shown that rosetting parasites form larger, stronger rosettes in non-O blood groups (A, B or AB) than in group O RBCs. Furthermore, the percentage of infected RBCs forming rosettes is significantly lower in fresh clinical isolates derived from group O than in non-O patients. It appears that this is because the A and B antigens are receptors for rosetting on uninfected RBCs, being bound by a parasite protein called PfEMP1 which is expressed on the surface of infected RBCs. Rosettes still form in group O RBCs (albeit smaller and weaker than in non-O RBC) through the involvement of other RBC molecules which act as alternative receptors for rosetting. Thus, it was reasoned that if rosetting contributes directly to the pathogenesis of severe malaria and is reduced in blood group O RBCs, then group O individuals should be protected against life-threatening malaria [6].

The life-saving effect of group O is thought to occur due to the impaired ability of Plasmodium falciparum parasites to form rosettes in group O blood. Rosetting occurs due to specific members of the P. falciparum Erythrocyte Membrane Protein one (PfEMP1) family of variant antigens, on the surface of infected red cells, binding to receptors, including the A and B blood group trisaccharides, on uninfected red cells. Rosetting contributes to microvascular obstruction in severe malaria, leading to hypoxia, acidosis, organ dysfunction and death [7].

In 2012, Vigan-Womas et. al. provided an insight to the mechanism explaining how rosetting malaria parasites bind to the blood group A sugars. The team used a comination of in vitro functional studies, insights from crystallography and computational docking studies to examine the binding site for interaction between the malaria parasite rosetting ligand, PfEMP1 and the group A-trisaccharide (GalNAc-α1,3(Fuc-α1,2)Gal). They expressed the extracellular domains from a rosette-mediating PfEMP1 variant (Palo Alto VarO) as recombinant proteins, and found that the N-terminal head-structure region (known as NTS-DBLα-CIDR), bound the group A-trisaccharide. Furthermore, they crystallized the PfEMP1 N-terminal region to analyze its structure, and used computer docking to identify potential binding sites for the A-trisaccharide [8].

The salient findings were:

1. VarO rosetting shares with other rosetting lines three generic characteristics, namely an extreme sensitivity to sulphated glycosaminoglycans, the need for human serum and a marked ABO blood group preference characterised by reduced binding to group O RBCs. 

2. The experimental data indicate that the major determinant affecting VarO rosetting efficiency is indeed the ABO blood group. 

3. On exploring VarO-infected RBCs (iRBCs) binding characteristics using a monovariant culture of the Palo Alto 89F5 clone, in which greater than 90% of the iRBCs were positively selected to express PfEMP1-VarO, it was observed that VarO-iRBCs preferentially bind to blood group A compared to blood group B, which itself is preferred to blood group O.

4. More detailed RBC subgroup analysis showed preferred binding to group A1, weaker binding to groups A2 and B, and least binding to groups Ax and O.

Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα1. The authors hypothesized that RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins. This opens novel opportunities for intervention against severe malaria.

In 2015, another aspect was explored to this ever expanding mechanism, the RIFINs [9]. Their presence has been reported as members of rif (repetitive interspersed family), clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum. Their high copy number, sequence variability, and red cell surface location indicated an important role for RIFINs in malaria host–parasite interaction but the mechanism was still missing [10]. The researchers now have attempted the same.

Though sequestration and rosetting in individuals with severe Plasmodium falciparum malaria has been attributed to P. falciparum erythrocyte membrane protein 1 (PfEMP1) still few questions remained:

a: Antibodies to PfEMP1 disrupt rosettes of parasites grown only in blood group O RBCs, not group A RBCs. 

b: The majority of P. falciparum strains and fresh clinical isolates prefer group A RBCs for rosetting. 

Researchers found that enzymatic removal of PfEMP1 from the iRBC surface reduced rosetting in blood group O but not blood group A, indicating that PfEMP1 may not be the only molecule responsible for RBC binding and rosette formation. A second family of antigens found at the iRBC surface is RIFINs. These polypeptides are encoded by 150 rif genes and comprise the largest family of antigenically variable molecules in P. falciparum. Given that the function of RIFINs is unknown and that they are resistant to enzyme degradation and upregulated in rosetting parasites, researchers speculated that RIFINs contribute to the rosetting and sequestration of P. falciparum mediated by blood group A antigen.

To study the function of the RIFINs, researchers investigated primary structures of RIFINs and found that the majority (~70%) belong to subgroup A (A-RIFIN) and possess an insertion of 25 amino acids at the N terminus (indel) that the B-RIFINs lack. Researchers analyzed the ability of rif gene–transfected CHO cells to bind RBCs. A-RIFIN CHO cells bound large numbers of group A RBCs (up to ~25 RBCs per CHO cell), whereas the binding of group O RBCs was less pronounced and similar to that of CHO cells expressing the N-terminal domain of PfEMP1 (DBL1α). This suggests that the group A antigen is a major receptor for A-RIFINs. RBC binding was negligible with B-RIFIN CHO cells or CHO cells expressing PfEMP1 (DBL1α of PfEMP1-FCR3S1.2var1) (control) [10]. Moreover, group A1 RBCs bound significantly better than group A2 RBCs. The researchers through various models examplified the role of RIFINs in microvascular binding of P. falciparum iRBCs. Their results hint that RIFINs may contribute to the varying global distribution of ABO blood groups in favor of blood group O.

1. Oliver-Gonzalez, J.; et. al. A substance in animal parasites related to the human isoagglutinogens. J Infect Dis 1944, 74, 173-177.
2. Raper, A. B. ABO blood groups and malaria. 1967.
3. Gupta, M.; et. al. Relationship between ABO blood groups and malaria. Bull World Health Organ 1980, 58(6), 913-915.
4. Singh, N.; et. al. ABO blood groups among malaria cases from district Mandla, Madhya Pradesh. Indian J Malariol 1995, 32(2), 59-63.
5. Carlson, J.; et. al. Plasmodium falciparum erythrocyte rosetting is mediated by promiscuous lectin-like interactions. J Exp Med 1992, 176(5), 1311-1317.
6. Rowe, J. A.; et. al. Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention. Curr Opin Hematol 2009, 16(6), 480-487.
7. Alexandra, R. J. Revealing the secrets of malaria parasite interaction with blood group A sugars. Pathog Glob Health 2013, 107(2), 45.
8. Vigan-Womas, I.; et. al. Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting. PLoS Pathog 2012, 8(7), e1002781.
9. Wahlgren, M.; et. al. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria. Nat Med 2015, 21(4), 314-317.
10. Kyes, S. A.; et. al. Rifins: A second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum. Proc Natl Acad Sci U S A 1999, 96(16), 9333-9338.

"Oliver like Einstein knew something, that we still dont"