How Protective Antibody Responses To Plasmodium Falciparum Drive Malaria Immunity
Antibodies targeting Plasmodium falciparum blood stage antigens play a critical role in malaria immunity. Learn how they help reduce disease severity.
Author:Suleman ShahReviewer:Han JuOct 14, 20246.1K Shares113.6K Views
The aim of this review was to discuss the identification of protective antibody responses to Plasmodium falciparum, specifically to its blood stage antigens.
Immunity to clinical disease and therefore, reduction of morbidity and mortality in Plasmodium falciparummalaria is largely mediated by immunity to antigens of blood stages.
People in hyperendemic areas often carry P. falciparumin their blood stream but remain asymptomatic, demonstrating:
- that there is a lack of development of natural immunity to infection
- the pre-erythrocytic stages of the parasite, including sporozoites and hepatic stages
Blood stages are merozoites, which are released from infected hepatocytes and red blood cells, and the intra-erythrocytic trophozoites and the schizonts.
Each merozoite exiting the liver into the bloodstream can invade an erythrocyte and multiply up to 20-fold every two days in cycles of erythrocyte invasion, replication, erythrocyte rupture, and release of infectious merozoites.
The erythrocyte rupture may release numerous P. falciparumblood stage antigens.
Objectives of this critical review were:
- to establish the theoretical foundations for identification of the pattern of antibody responses associated with protection against clinical manifestations of severe P. falciparummalaria
- to establish a pathway leading to the discovery of immuno-relevant blood stage antigens of P. falciparum, which could form the basis of future vaccines against blood stages of this parasite
Specific Antibody Subclass Responses And Immunity To Clinical Malaria
The first experiments demonstrating the proof of principle of the role of antibodies in anti-plasmodial immunity in humans were gamma-globulin transfer experiments from immune Gambian adults to children with asymptomatic P. falciparumparasitemia, resulting in a:
- reduction to the parasite count to <1% of the initial value
- progressive reduction of clinical symptoms
Later studies showed that global immunoglobulin responses against P. falciparumwere not associated with protection but immunoglobulin (Ig) subclasses IgG1 and IgG3.
In French Guinea, a cross-sectional study investigated the levels of antibodies comparing persons living in endemic areas without parasitemia and persons from endemic areas with acute malaria.
Parasitemia was associated with lower IgG1 and IgG3 subclass responses.
In termsof protective antibody responses to blood stage antigens previously investigated in immune-epidemiological studies, a recent systematic review with meta-analysis of population-based prospective studies and population-based treatment to reinfection studies found a limited number of studies and restricted to antigens/components of antigens comprising:
| MSP-119 | merozoite surface protein-119 |
| MSP-1-EGF | merozoite surface protein-1-epidermal growth factor |
| MSP-1-BL1 | merozoite surface protein-1-BL1 |
| MSP-1-BL2 | merozoite surface protein-1-BL2 |
| MSP-2 | merozoite surface protein-2 |
| MSP-2 ACD | merozoite surface protein-2 accidental cell death |
| MSP-3 | merozoite surface protein-3 |
| GLURP | glutamate-rich protein |
| AMA-1 | apical membrane antigen 1 |
| EBA-175 | erythrocyte binding antigen 175 |
The authors concluded that IgG (immunoglobulin G)responses to some, but not all, merozoite surface antigens were associated with protection against symptomatic P. falciparuminfection in malaria endemic areas.
The authors identified very few antigens that had been well studied and a deficiency of studies done outside Africa.
The authors concluded that more studies in different populations, examining multiple antigens at multiple time-points, are needed to better determine the role of anti-merozoite antibodies in protection against malaria, with prospective cohort studies as the preferred study design to establish temporal causality.
Detection Of Immune-Relevant Antigens
Immunoglobulin subclasses IgG1 and/or IgG3 were associated with protection.
This protection is provided by the ability of these subclasses to link antigens to monocytes stimulating them to produce tumor necrosis factor.
This cytokine induces production of nitric oxide, which kills intracellular parasites as part of antibody dependent cellular inhibition (ADCI) and the generated nitric oxide is a vasodilator preventing cerebral vasospasm, which is significantly involved in the pathogenesis of cerebral malaria.
IgG1 and IgG3 were therefore called cytophilic or opsonizing antibody subclasses.
The initial investigations of antibody responses have mainly been carried out by ELISA (enzyme-linked immunosorbent assay)using recombinant proteins or synthetic peptides usually representing subdomains of malarial proteins as test antigens.
Such antigen preparations don’t reflect native parasite protein conformations.
The first study using an unbiased approach to detection of immunoreactive antigens visualized antigens, which were precipitated by immunoglobulin of immune donors.
This was achieved by labeling the parasite with 35S-methionine and separating the immune-complexes by two-dimensional electrophoresis followed by auto-radiographic detection of the position of the immune-complexes in relation to molecular weight standards.
This method did not allow identification of the antigens, immunoglobulin subclasses, or avidity.
The method could also not exclude missing small antigens trapped in the immune-precipitants, which were not bound by immunoglobulins. This is particularly important for low abundance antigens.
The first study to enable a description of the antibody type as well as the molecular mass of antigens in people exposed to P. falciparummalaria used one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with 21 molecular weight markers followed by electrotransfer to a nitrocellulose paper.
Antibody types and IgG subclasses were visualized and 38 detergent solubilized parasite antigens and 20 exoantigens were visualized without facility to identify these antigens or correlate them to clinical immunity.
The immunoblotting patterns indicated that immune adult sera of the investigated people from Burkina Faso contained antibodies of all IgG subclasses specific for the whole series of somatic antigens.
IgG1 was predominant and IgG4 was the least prevalent subclass.
Further progress in identification of immunoglobulin subclass responses to native P. falciparumblood stage antigens, correlating with clinical immunity, was also achieved by one-dimensional SDS-PAGE and immune-blotting.
People with different exposure to P. falciparumwere investigated.
There was a tendency for a broader IgG1 and IgG3 reactivity with increased exposure.
Non-immune Danish travelers with a single malaria episode reacted against a few high-molecular weight antigens, whereas those from Liberian adults recognized a larger number of both high-molecular and low-molecular weight antigens.
The authors concluded that, the long time required to acquire clinical protection against P. falciparummalaria is not only related to isotype switching towards ADCI (antibody dependent cellular inhibition)-effective antigens but also to a gradual development of IgG1 and IgG3 antibodies against some previously non-targeted antigens.
This approach did not identify the antigens involved, but was a less biased approach compared to protein microarray methods using recombinant proteins or synthetic peptides being developed.
The most comprehensive approach to detection of antigens relevant in a protective immunoglobulin response was pioneered by two studies both published in 1999 by the journal Electrophoresis:
- one has Aida Pitarch as lead author
- the other one has Peter R. Jungblut, Gertrud Grabher, and Georg Stöffler as authors
Jungblut et al. and Pitarch et al. subjected native proteins of Borrelia garinii(bacteria) and Candida albicans(fungi), respectively, to two-dimensional electrophoresis followed by Western blotting with serum of patients.
This approach was first successfully applied to parasites with a more complex proteome for the nematode Trichostrongylus colubriformisin a later study by a different group.
An approach to identification of immunorelevant antigens of a protective immune response to P. falciparumcould be as follows (modified from Michael Eisenhut’s study published in 2010 by the journal Autoimmunity):
a. Antigens against a wide spectrum of P. falciparumantigens could be gained from purified parasitophorous vacuole membrane-enclosed merozoite structures (PEMS), which contain a highly homogeneous synchronous parasite population at the mature schizont stage, which is essentially free of contaminating host cell proteins.
b. Antigen is prepared by saponin mediated lysis of parasitized red blood cells followed by washing steps, and breaking up of parasites by trituration and ultrasonication.
c. The antigen mixture is subjected to two-dimensional electrophoresis using isoelectric focusing in the first using a pH gradient of 2 to 12 in the IPG (immobilized pH gradient) strip and PAGE (polyacrylamide gel electrophoresis) in the second dimension. Four gels are generated for each participant.
d. Antigens are blotted onto nitrocellulose where they get exposed to serum of a person with and separately without clinical immunity to P. falciparum.
e. For identification of high avidity antibodies, the blotted antigen-antibody complexes on two of the four nitrocellulose sheets per participant are subjected to a washing step, with a dissociation buffer including PBS-T-urea (8M) with vigorous shaking, followed by additional wash with PBS-T (phosphate-buffered saline with Tween 20) buffer.
f. Bound immunoglobulin is visualized with anti-human immunoglobulin. Immunoglobulin subclasses are visualized by specific anti-subclass antibodies for the cytophilic antibody subclasses IgG1 and IgG3 conjugated with peroxidase. One set of two gels (one with and one without dissociation buffer treatment) is used for each antibody subclass.
g. The amount of antibody of the participant’s serum bound to the antigen is quantified by transmission densitometry of the stains generated by the peroxidase reaction.
h. Avidity is quantified by the avidity index as ratio of the optical density of urea-treated spots to the optical density value of untreated spots multiplied by 100. An avidity index of less than 30% is defined as low-avidity, between 30% and 50% as intermediate avidity, and greater than 50% as high-avidity.
i. The patterns emerging with this method are put into relation to clinical immunity. Groups with clinical immunity are compared to patients hospitalized with acute malaria. Subgroup analysis is done for different age groups.
j. Antigens found to be associated with a protective antibody response in prospective studies can be identified by mass spectrometry using sections of the gel where the antigen is situated. Amino acid sequences found by this process can be attributed to proteins using the fully deciphered P. falciparumgenome.
Discussion
The foundation of development of effective blood stage vaccines is comprehensive identification of protective antibody responses.
These are characterized by cytophilic properties (i.e., IgG1 and IgG3) and high avidity and directed against multiple antigens.
Preliminary studies suggest that, in the context of host immune recognition of the P. falciparumparasite, a large number of antigens are recognized, dispersed amongst a large fraction of the proteome.
A currently pursued approach to detection of antigens relevant for immunity to P. falciparumis the detection of genetic signatures of balancing selection to identify targets of anti-parasite immunity.
Balancing selection is selection that maintains different alleles within a population.
Using this approach, a variety of given polymorphisms of genes is maintained rather than a reduction of numbers of gene variants by selection or persistence of a random neutral variety of polymorphisms.
These gene sequences undergoing balancing selection can be identified by sequencing and comparison of the sequences obtained.
Disadvantages include that over 5,000 genes will have to be screened and epigenetic modification is not captured by this method. Another shortcoming difficult to overcome with this method is expansion of the population and heterogeneityof the population sampled.
The starting point for identification of effective antibody responses requires a detailed and comprehensive description of the “immunome” defined as the set of antigens or epitopes that interface with the host immune system.
The above detailed approach of two-dimensional PAGE followed by immunoblotting is unbiased compared to analysis of antibody responses to selected recombinant P. falciparumantigens on high-throughput microarrays and macroarrays.
Recombinant proteins lack epigenetic modifications and glycosylation and may not have epitope forming tertiary and quaternary structures; for example, those formed by disulfide bonds.
Two-dimensional electrophoresis as an approach also has limitations: protein surface structure may be altered by detergent use.
Hydrophobic proteins may not be released from their position in parasite or red blood cell membranes or fail to migrate in the gel.
The resolution of the two-dimensional electrophoresis may not be high enough to enable detection of low abundance proteins, with the wide range of the pH gradient used to capture a comprehensive set of blood stage antigens.
An additional agenda in research investigating protective antibody responses is looking for antigens inducing antibodies with adverse effects on the clinical manifestations of malaria: IgG antibody responses to conserved regions of merozoite proteins and immune-complex concentrations were found to be elevated in patients with cerebral malaria compared to uncomplicated malaria.
This may be partly due to down-regulation of Fc-receptors (FcRs)involved in the clearance of antibodies but could indicate a pathogenetic role of immune-complexes particularly of those containing IgE.
Deposition of IgE containing immune-complexes could lead to the release of TNF (tumor necrosis factor), a cytokine that leads to increased cytoadherence of parasitized red blood cells by up-regulation of ICAM 1 (intercellular adhesion molecule-1) in endothelial cells.
P. falciparummalaria is commonly associated with features of auto-immune glomerulonephritis and future studies need to explore the specificity and antibody subclass of the IgG involved to avoid adverse effects of vaccine induced antibody responses.
Conclusion
Future research needs to employ the unbiased platform of two-dimensional electrophoresis combined with immunoblotting onto nitrocellulose and detection of high avidity and cytophilic antibody responses in sera of clinically immune compared to patients with severe malaria with identification of the immune-relevant antigens detected by mass spectrometry.
This will enable development of a multi-epitope vaccine with a higher rate of protection.
In addition, new information about antibody responses to Plasmodium falciparumcan be gathered from future research.
Suleman Shah
Author
Suleman Shah is a researcher and freelance writer. As a researcher, he has worked with MNS University of Agriculture, Multan (Pakistan) and Texas A & M University (USA). He regularly writes science articles and blogs for science news website immersse.com and open access publishers OA Publishing London and Scientific Times. He loves to keep himself updated on scientific developments and convert these developments into everyday language to update the readers about the developments in the scientific era. His primary research focus is Plant sciences, and he contributed to this field by publishing his research in scientific journals and presenting his work at many Conferences.
Shah graduated from the University of Agriculture Faisalabad (Pakistan) and started his professional carrier with Jaffer Agro Services and later with the Agriculture Department of the Government of Pakistan. His research interest compelled and attracted him to proceed with his carrier in Plant sciences research. So, he started his Ph.D. in Soil Science at MNS University of Agriculture Multan (Pakistan). Later, he started working as a visiting scholar with Texas A&M University (USA).
Shah’s experience with big Open Excess publishers like Springers, Frontiers, MDPI, etc., testified to his belief in Open Access as a barrier-removing mechanism between researchers and the readers of their research. Shah believes that Open Access is revolutionizing the publication process and benefitting research in all fields.
Han Ju
Reviewer
Hello! I'm Han Ju, the heart behind World Wide Journals. My life is a unique tapestry woven from the threads of news, spirituality, and science, enriched by melodies from my guitar. Raised amidst tales of the ancient and the arcane, I developed a keen eye for the stories that truly matter. Through my work, I seek to bridge the seen with the unseen, marrying the rigor of science with the depth of spirituality.
Each article at World Wide Journals is a piece of this ongoing quest, blending analysis with personal reflection. Whether exploring quantum frontiers or strumming chords under the stars, my aim is to inspire and provoke thought, inviting you into a world where every discovery is a note in the grand symphony of existence.
Welcome aboard this journey of insight and exploration, where curiosity leads and music guides.
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