Malaria: An Exploration of Plasmodium Visibility and the Antibody Response
Introduction
Malaria is a vector-borne disease caused by the parasitic protist Plasmodium Falciparum. It is among the leading causes of death worldwide: 3.2 million people are at risk of infection annually, with an estimated 200-500 million people infected and 600,000-one million reported annual deaths (1,6). The brunt of these infections occur in poor, provincial areas near the equator and in sub-saharan Africa, where heavy rainfall and inadequate infrastructure create a hospitable environment for the disease vector: the anopheles mosquito (16). The mosquito lays its eggs in standing water; everything from a lake or pond to a bucket left out in the rain can bring a disease vector into one’s home or community. The anopheles salivary glands become infected when it takes a blood meal from an infected mammal. Plasmodium matures in the salivary glands, and can be transferred to a new host when the mosquito again takes a blood meal (6). While mosquito eradication and bite-avoidance techniques hold sway as practical means of malaria suppression, inventing a viable vaccine has long been a holy grail of the immunology and global health communities. But whereas exposure to many pathogens–like those causing measles and smallpox–confers long lasting, sterile immunity to infected individuals, plasmodium infection has been shown to cause only short-lived memory immune responses, with clinical immunity developing only after years of repeated exposure (3,1). This quality has made vaccine development a continuing challenge. In this review, I will discuss plasmodium in terms of the various windows during which the pathogen is visible to the immune system; the importance of antibodies in mounting a humoral immune response; and finally the antigenic plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP1)–a protein that has revealed much about natural acquisition of clinical immunity.
To understand why a malaria vaccine has eluded researchers for so long, it is important to understand the mechanism of plasmodium pathogenesis, and how the human immune system encounters the pathogen during different parts of its life cycle. During the course of infection, Plasmodium inhabits three different parts of the human body and goes through two distinct phases of growth and reproduction.
In the Liver–The Pre-erythrocytic Cycle
In the pre-erythrocytic cycle, the pathogen enters the bloodstream as a sporozoite, and makes its way to the liver (4,6,8,18). Some evidence suggests that sporozoites access the liver through an existing host clearance system. For example, Sinnis at al. have shown that the plasmodium circumsporozoite (CS) protein competes with degraded lipoproteins for clearance to the liver by cells that normally carry cellular detritus (18). This is a brief window during which time the entire pathogen is exposed to encounter with immune cells. Within a matter of minutes after a mosquito bite, the sporozoites access liver blood vessels, where membrane-membrane contact allows the pathogens to enter into Kupffer cells (KCs) (11). Inside KCs, sporozoites enter a vacuole that does not fuse with lysosomes, thereby avoiding degradation (11). The CS protein, along with the thrombospodin-related adhesive protein (TRAP)–another surface protein–mediate invasion of the space of Disse, and entrance into liver cells (8,18).
As the liver is in constant contact with toxins, allergens, pathogens, and other inflammation causing agents, it exhibits inherent immune tolerance so as to avoid a self destructive immune response (8,11). This tolerance is achieved via three main cellular strategies. First, several classes of liver antigen-presenting cells (APCs), including KCs, express low levels of MHC class I and II and co-stimulatory surface molecules (11). Second, liver APCs excrete anti-inflammatory cytokines, such as IL-10 and TGF𝛃, and there is evidence of basal clonal T-cell deletion (11). Lastly, through ill-understood intracellular mechanisms, plasmodium has been shown to downregulate MHC and IL-12 (inflammatory cytokine) production, and also to upregulate IL-10 production inside innate liver leukocytes.
Even those lymphocytes that do activate in reaction to liver-stage plasmodium infection can only recognize antigens like CS protein and TRAP that cease to be expressed during the blood stage, and cannot recognize the pathogen after it leaves the liver. All of this allows the sporozoites to easily avoid detection by the host immune system during the liver phase, and to largely prevent activation of plasmodium-antigen-specific naive B-cells or CD8 T-cells (8,11,18). As such, liver stage antigens are not thought to contribute much to acquisition of clinical immunity.
Once inside liver cells, plasmodium matures into schizonts, a rapidly dividing form of the pathogen. Safe from immune detection, the schizonts multiply and mature into the pathogen’s blood stage. This process involves expression of novel protein antigens (11). Schizonts typically hit a critical mass of 10,000-30,000 organisms inside an infected liver cell before the cell ruptures, releasing an army of pathogens back into the bloodstream (8). Once released into the blood, plasmodium reaches its merezoite phase (6).
In the Blood–The Erythrocytic Cycle
Once in the bloodstream, merezoites infect red blood cells (RBCs). Invasion of RBCs involves a weak-attachment and strong-attachment steps, mediated by several conserved surface proteins (14). Some, such as P. falciparum reticulocyte-binding homologues (PfRHs) and erythrocyte binding antigens (EBAs), have been identified as important antigens in the immune response to plasmodium infection (20). These proteins form a tight junction between merezoites and red blood cells, and position the apical pole of the pathogen for entry into the erythrocyte (4).
Similar to initial sporozoite infection, merezoites in the bloodstream provide another brief window wherein the entire pathogen is visible to the immune system. While antibodies to bloodstream merezoites have been shown to develop, they are not thought to confer protection from severe disease (14,8). The only phase of the plasmodium life cycle during which antigens are consistently visible to lymphocytes occurs after the pathogen has infected host erythrocytes.
Once inside the erythrocyte, plasmodium matures into a trophozoite–the stage of the plasmodium life cycle associated with clinical symptoms (6,14,8). Trophozoites can mature in one of two ways: they can proceed to an asexuel schizont phase in which rapid replication leads to RBC rupture and increased parasitemia; or mature into gametocytes capable of infecting new mosquitos (6). The trophozoites that continue to multiply and infect RBCs begin to express novel proteins that cause RBCs to sequester in the bloodstream. This helps RBCs avoid clearance to the spleen (14,5). The P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1) is one such protein.
PfEMP1 is a protein that protrudes from the outside of infected erythrocytes. It allows erythrocytes to become stuck in the bloodstream. PfEMP1 actually comprises a highly variant family of segmented proteins, consisting of a chain of different PfEMP1 domains. The protein is coded for by the var genes–a ~60 gene haplotype that is differentially expressed in isogenic organisms. Each different var gene encodes a different PfEMP1 domain, with each domain binding a different vascular surface protein. Protein recombination means that infection by isogenic pathogens can contain all ~60 PfEMP1 phenotypes.
Domains are split into two different types: Duffy-binding Like (DBL) and Cysteine-rich Inter-Domain Region (CIDR), which are subdivided further based on sequence similarity (5). Many host proteins that PfEMP1 binds to are located in the vascular endothelium, allowing those RBCs to sequester in the bloodstream. These include CD36 and the Erythrocyte Protein-C Receptor (EPCR) (5). However, some bind to host proteins elsewhere in the body. PfEMP1 domains binding ICAM1, for example, have been associated with cerebral malaria, and domains binding chondroitin sulfate A have been shown to sequester in the placenta, leading to pregnancy-associated malaria (4). Additionally, some of these PfEMP1 domains cause erythrocyte rosetting–the attachment of uninfected erythrocytes to an infected erythrocyte (4,5,14). This class of proteins is currently a focal point in malaria immunity research.
In summary, immune responses to plasmodium infection become jumbled by changing antigen expression and progressive infection of different parts of the body. While antigens are recognized at various stages of the plasmodium lifecycle, PfEMP1 is one of very few pathogenic proteins that has been shown to confer immunity.
Antibody Response
Antibodies to the various domains of PfEMP1 are thought to be essential in the development of clinical immunity to malaria (4,3). They prevent PfEMP1 from binding host endothelial receptors, from mediating rosetting, and increase opsonic phagocytosis (14). One area of promising new research is the identification of particular PfEMP1 binding domains associated with severe disease, and subsequent testing of how antibodies to those domains affect disease symptoms.
In a regent longitudinal study conducted in Tanzania, Turner et al. (2015) sought to characterize the complex mechanism of anti-PfEMP1 antibody development (5). A previous study lead by Turner had identified several CIDR domains that display high affinity binding to EPCR, as well as a positive correlation between plasmodium parasites with those EPCR-binding CIDR phenotypes and severe malaria (21). In the 2015 study, patients of all ages across malaria endemic and malaria non-endemic regions were tested for the presence of anti-EPCR-binding CIDR IgG antibodies in their blood. In the malaria endemic regions, the antibody was found to develop earlier in life than other anti-PfEMP1 antibodies, and to increase in concentration between age 0 and 11, subsequently decreasing through age 60. Furthermore, concentration of the antibody was found to drop off when malaria transmission rates were inconsistent. Despite not testing this themselves, “combined evidence” suggests that anti-EPCR-binding CIDR antibodies do confer protective immunity against a certain PfEMP1 phenotype. But they also claim that effective targeting of one PfEMP1 phenotype can push proliferation of other PfEMP1 phenotypes in that individual (5). Taken as a whole, their results indicate that protective antibodies to PfEMP1 do develop in response to plasmodium infection. However, these antibodies cannot persist without continuous antigenic exposure, nor can they protect from infection by pathogens expressing different PfEMP1 phenotypes. Other studies have similarly shown that antibodies appear to always be a step behind the pathogen (2,8,10,13).
One reason for imperfect antibody development may be that plasmodium infection can impair B-cell-mediated immunity (13). Recall that antibodies develop as surface proteins on activated B-cells. B-Cells differentiate in the bone marrow, and develop B-Cell Receptors (BCRs) that are highly specific to non-self antigens. Once mature, B-cells migrate to secondary lymphoid organs, where they can become activated through binding to a specific antigen, in addition to, in many cases, T-cell co-stimulation. Once activated, B-cells proliferate, and undergo different processes to maximize specificity of the BCRs to a particular antigen. One of these processes occurs in a microenvironment called the Germinal Center (GC), where follicular helper T-cells (Tfh) mediate development of highly specific BCRs (22). In a 2015 study, Ryg-Cornejo et al. found that Plasmodium infection inhibits maturation of Tfh cells. Mouse Tfh cells displayed low levels of PD-1 and CXCR5–Tfh associated proteins–as well as continued expression of T-bet and CXCR3–proteins characteristic of undifferentiated helper T-cells. This resulted in reduced formation of germinal centers (13). Several other researchers, such at Hviid (2014), White (2014), and Scholzen (2013), have similarly shown that B-cell function is impaired with plasmodium infection (3,10,13).
Despite this, research has shown that clinical immunity does develop after years of repeated exposure, likely due to a buildup of different antibody responses to different antigens at various phases of the plasmodium life cycle (1,2,3.10,13,23,24).
Conclusion
The humoral response to the malaria pathogen is shaped by the different windows of visibility and invisibility inherent to the plasmodium life cycle. As plasmodium travels between the bloodstream, the liver, and Red Blood Cells, it expresses different sets of antigens, with further different subsets of those antigens visible to the immune system. This results in a highly complex, lengthy process of developing adaptive immunity against malaria.
Additionally, the complex immune response to malaria has made vaccine development difficult. Current avenues of vaccine research are looking into liver-stage antigens, blood-stage antigens (including PfEMP1), as well as combinations of the two (for a comprehensive look at past, present, and intended future vaccine research, see Outtara and Laurens’ 2014 review, “Vaccines Against Malaria”) (25). However, much is yet understudied. Future research should focus on profiling the set of antibodies present in clinically immune individuals to better understand what combinations of antibody responses can prevent severe disease.
Sources:
- Weiss GE, Traore B, Kayentao K, Ongoiba A, Doumbo S, et al. (2010) The Plasmodium falciparum-Specific Human Memory B Cell Compartment Expands Gradually with Repeated Malaria Infections. PLoS Pathog 6(5): e1000912. doi:10.1371/journal.ppat.1000912
- Dodoo, D., et al. “Antibodies to Variant Antigens on the Surfaces of Infected Erythrocytes Are Associated with Protection from Malaria in Ghanaian Children.” Infection and Immunity, vol. 69, no. 6, Jan. 2001, pp. 3713–3718. NCBI, doi:10.1128/iai.69.6.3713-3718.2001.
- Scholzen, Anja, and Robert W. Sauerwein. “How malaria modulates memory: activation and dysregulation of B cells in Plasmodium infection.” Trends in Parasitology, vol. 29, no. 5, 2013, pp. 252–262., doi:10.1016/j.pt.2013.03.002.
- Lavstsen T, Salanti A, Jensen AT, Arnot DE, Theander TG. 2003. Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions. Malar J 2:27.
- Turner, Louise, et al. “IgG Antibodies to Endothelial Protein C Receptor-Binding Cysteine-Rich Interdomain Region Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 Are Acquired Early in Life in Individuals Exposed to Malaria.” Infection and Immunity, vol. 83, no. 8, 2015, pp. 3096–3103., doi:10.1128/iai.00271-15.
- “Malaria.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 18 Oct. 2017, www.cdc.gov/malaria/.
- “Biology.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 1 Mar. 2016, www.cdc.gov/malaria/about/biology/index.html.
- Bertolino, Patrick, and David G. Bowen. “Malaria and the liver: immunological hide-and-Seek or subversion of immunity from within?” Frontiers in Microbiology, vol. 6, 2015, doi:10.3389/fmicb.2015.00041.
- Epstein, Judith E., and Thomas L. Richie. “The whole parasite, pre-Erythrocytic stage approach to malaria vaccine development.” Current Opinion in Infectious Diseases, 2013, p. 1., doi:10.1097/qco.0000000000000002.
- Hviid, Lars, et al. “Trying to remember: immunological B cell memory to malaria.” Trends in Parasitology, vol. 31, no. 3, 1 Mar. 2015, pp. 89–94., doi:10.1016/j.pt.2014.12.009.
- Krzych, Urszula, et al. “Memory CD8 T Cells Specific for Plasmodia Liver-Stage Antigens Maintain Protracted Protection Against Malaria.” Frontiers in Immunology, vol. 3, Oct. 2012, doi:10.3389/fimmu.2012.00370.
- Linterman, Michelle. “Faculty of 1000 evaluation for Circulating Th1-Cell-Type Tfh Cells that Exhibit Impaired B Cell Help Are Preferentially Activated during Acute Malaria in Children.” F1000 – Post-Publication peer review of the biomedical literature, May 2016, doi:10.3410/f.725829687.793512836.
- Ryg-Cornejo, Victoria, et al. “Severe Malaria Infections Impair Germinal Center Responses by Inhibiting T Follicular Helper Cell Differentiation.” Cell Reports, vol. 14, no. 1, 5 Jan. 2016, pp. 68–81., doi:10.1016/j.celrep.2015.12.006.
- Teo, Andrew, et al. “Functional Antibodies and Protection against Blood-Stage Malaria.” Trends in Parasitology, vol. 32, no. 11, 1 Nov. 2016, pp. 887–898. ScienceDirect, doi:10.1016/j.pt.2016.07.003.
- Voza, Tatiana, et al. “Intradermal immunization of mice with radiation-Attenuated sporozoites of Plasmodium yoelii induces effective protective immunity.” Malaria Journal, vol. 9, no. 1, 15 Apr. 2010, p. 362., doi:10.1186/1475-2875-9-362.
- N. Obeng-Adjei, S. Portugal, T.M. Tran, T.B. Yazew, J. Skinner, S. Li, A.Jain, P.L. Felgner, O.K. Doumbo, K. Kayentao, et al. “Circulating Th1-cell-type Tfh cells that exhibit impaired B cell help are preferentially activated during acute malaria in children.” Cell Rep., 13 (2015), pp. 425-439
- “Tropical Medicine & International Health.” Tropical Medicine & International Health, vol. 22, no. 6, 2017, pp. 655–655., doi:10.1111/tmi.12891.
- Pradel G., Garapaty S., Frevert U. (2002). Proteoglycans mediate malaria sporozoite targeting to the liver. Mol. Microbiol. 45, 637–651. 10.1046/j.1365-2958.2002.03057.x
- Seo, Hyo Jung, et al. “Rapid Hepatobiliary Excretion of Micelle-Encapsulated/Radiolabeled Upconverting Nanoparticles as an Integrated Form.” Scientific Reports, vol. 5, no. 1, 2015, doi:10.1038/srep15685.
- Persson, K.E. et al. (2008) Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies. J. Clin. Invest. 118, 342–351
- Turner L, Lavstsen T, Berger SS, Wang CW, Petersen JE, Avril M, Brazier AJ, Freeth J, Jespersen JS, Nielsen MA, Magistrado P, Lusingu J, Smith JD, Higgins MK, Theander TG. 2013. Severe malaria is associated with parasite binding to endothelial protein C receptor. Nature 498: 502–505.
- Murphy, Kenneth, and Casey Weaver. Janeways immunobiology. Garland Science/Taylor & Francis Group, LLC, 2016.
- White, M.T. et al. (2014) Dynamics of the antibody response to Plasmodium falciparum infection in African children. J. Infect. Dis. 210, 1115–1122.
- Zander, Ryan A., et al. “Th1-Like Plasmodium -Specific Memory CD4 T Cells Support Humoral Immunity.” Cell Reports, vol. 21, no. 7, 14 Nov. 2017, pp. 1839–1852., doi:10.1016/j.celrep.2017.10.077.
- Ouattara, A., and M. B. Laurens. “Vaccines Against Malaria.” Clinical Infectious Diseases, vol. 60, no. 6, Jan. 2014, pp. 930–936., doi:10.1093/cid/ciu954.