Characterization of a Pfemp1 variant from a P. falciparum field is isolate and the development of anti-Pfemp1 antibodies

Rosetting (binding of infected erythrocytes to uninfected ones) is a parasite adhesion phenotype thought to be mediated by Plasmodium falciparum membrane protein 1 (PfEMP1). Despite the association between rosetting and severe malaria in Africa, rosette-mediating PfEMP1 variants from African-derived strains are yet to be characterized. I initially attempted to detect the predominantly expressed var genes in five recently culture-adapted Kenyan P. falciparum isolates selected for rosetting. Unfortunately no clear rosetting-associated genes could be detected, possibly because of incomplete selection and lack of a clear phenotype in the selected parasites. I then went on to study another Kenyan parasite line, SA075, whose predominant var gene tag had interesting sequence features previously linked to rosetting. Using PCR walking, cloning and sequencing, I identified the full-length var gene corresponding to this tag, called SA075var1. Subsequent experiments failed to confirm a role for the PfEMP1 variant encoded by SA075var1 in rosetting. However, SA075var1 had domain cassette DC 8-like features which have lately been linked to severe disease. Antibodies raised in rabbits against recombinant proteins from domains of SA075var1 revealed functional antibodies with an ability to recognize the surface of parasite infected erthrocytes and to mediate phagocytosis. Although mainly variant-specific, the antibodies showed limited cross-reactivity with one other parasite strain. Using plasma pairs from Kenyan malaria patients, I examined antibody responses against SA075 and four other rosetting P. falciparum strains selected to enrich for single rosette-mediating variants. Antibody responses were seen against particular rosette-elected strains suggesting that some variants may be important for particular severe malaria syndromes. Overall, the data described in this thesis demonstrates the need for continued research into functional and immunological characterization of PfEMP1 variants, especially in field isolates. Antibody data however showed clinical relevance of some rosette-mediating variants that could become important targets for malaria intervention studies

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal

Impact of RTS,S/AS02A and RTS,S/AS01B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

Objective:RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals\u27 proprietary Adjuvant Systems AS02A or AS01B. A recent trial of the RTS,S/AS02A and RTS,S/AS01B vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02A and RTS,S/AS01B on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.Design:The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.Setting:The study was conducted in Kombewa District, western Kenya.Participants:Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.Outcome:Parasite isolates used for determining MOI and divergence of csp T cell&ndash;epitopes were 191 at baseline and 87 from break-through infections.Results:Grouping recipients of RTS,S/AS01A and RTS,S/AS02B together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01B group, but not the RTS,S/AS02A group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.Conclusions:It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct

Females of HbAS genotype have reduced concentration of the malaria protective deoxyhemoglobin S than males.

The quantity of the intra-erythrocytic deoxyhemoglobin S (Hb S) affects the level of protection against malaria and also the sickling phenomenon. This study reports on significantly lower concentration of Hb S in females than males. Data came from 350 children, aged 12-47 months who participated in a phase 2b malaria vaccine trial. Hemoglobinopathy and G6PD deficiency typing was necessary to ascertain equal representation of these malaria protective traits across the vaccine cohorts. Hemoglobin types (HbAA, HbAS) and % Hb S were evaluated by HPLC. Alpha thalassemia (alpha-thal) and G6PD genotypes were evaluated by PCR. The overall prevalence for HbAS was 20%, 46% for 3 alpha genes and 10% for 2 alpha genes and 14% for G6PD A-. More females of HbAS/αα/αα genotype had low Hb S than males and had mean % Hb S of 37.5% ± 5.4 SD, compared to 42.0% ± 2.5 SD in males of same genotype (P = 0.018). Consistent with reduction of the malaria protective Hb S in females, parasite load in females was nearly twice that of males but the difference was not statistically significant. The X-chromosome linked G6PD deficiency did not influence the level of Hb S. We conclude that, the low Hb S in these females explains the resultant higher malaria parasite load. We speculate that the low Hb S in females could also explain observations suggesting that the sickling phenomenon tends to be less severe in females than males

Enzyme-Linked Immunosorbent Assay for Detection of Plasmodium falciparum Histidine-Rich Protein 2 in Blood, Plasma, and Serum▿

Microscopy, the gold standard for the detection and quantification of malaria parasites in blood, is in many aspects deficient for this purpose. The method is poorly reproducible and can be inaccurate because Plasmodium falciparum parasites sequester for a portion of each asexual cycle. Due to these deficiencies, biomarkers such as P. falciparum histidine-rich protein 2 (PfHRP2) are increasingly being used. In this study, we evaluated the use of a commercial PfHRP2 enzyme-linked immunosorbent assay (ELISA) kit with some procedural modifications. We determined the linear range of the assay, including the lower limits of detection and quantitation, using recombinant PfHRP2 (rPfHRP2). In 10 repeat experiments, the linear range of optical densities (ODs) at 450 to 650 nm was from 0.05 ± 0.002 to 2.28 ± 0.042, corresponding to 3.91 to 250 ng/ml of rPfHRP2. The coefficient of variation (CV) at each target concentration ranged from 1.93 to 8.07%. Using cultured parasites, we confirmed the linear range of ODs as well as the association between the PfHRP2 ELISA results and the microscopic parasite densities. For whole-blood samples spiked with cultured, washed, ring-stage-infected red blood cells (iRBCs), the linear range was 11.7 to 750 iRBCs/μl, with CVs of 0.29 to 7.56%. The same spiked samples evaluated by microscopists had similar sensitivities, but the CVs were unacceptably high (20.7 to 161.6%). Stock rPfHRP2 was stable through four freeze-thaw cycles (P < 0.05; paired t test). When different patient sample types at different concentrations within the linear range of the assay are compared, the recoveries of PfHRP2 from blood and serum were within ±20%, whereas the recoveries from plasma ranged between +35 and −41%. We conclude that PfHRP2 ELISA using whole-blood and serum samples is a suitable adjunct to microscopy and could ultimately benefit malaria intervention trials

DataSheet_1_Longitudinal impact of asymptomatic malaria/HIV-1 co-infection on Plasmodium falciparum gametocyte transcript expression and transmission to Anopheles mosquitoes.docx

Despite significant developments towards malaria reduction, parasite transmission in the common context of HIV-1 co-infection and treatment for one or both infections has not been fully characterized. This is particularly important given that HIV-1 and malaria chemotherapies have the potential to alter gametocyte burden and mosquito infectivity. In this study, we examined 782 blood samples collected from a longitudinal cohort of 300 volunteers with asymptomatic parasitemia seeking HIV testing or treatment in the endemic region of Kisumu, Kenya, to define the impacts of HIV-1-malaria co-infection, antiretroviral therapy (ART) plus trimethoprim-sulfamethoxazole (TS) and the antimalarials artemether/lumefantrine (AL) on Plasmodium falciparum gametocyte transcript prevalence and parasite transmission to the African malaria mosquito Anopheles gambiae. Volunteers were assigned to three distinct HIV-1 groups: HIV-1 positive on treatment, HIV-1 positive newly diagnosed, and HIV-1 negative. Volunteers were monitored monthly over the course of six months. Using our highly sensitive digital droplet PCR (ddPCR) assay of three gametocyte specific transcript markers, we detected gametocyte transcripts in 51.1% of 18S positive volunteers across all study groups and time points. After correcting for multiple comparisons, the factors of HIV-1 status, time, CD4+ T-cell levels and hematocrit were not predictive of gametocyte prevalence or transmission. However, among those volunteers who were newly diagnosed with HIV-1 and malaria positive by rapid diagnostic test (RDT) at enrollment, the initiation of ART/TS and AL treatment was associated with a significant reduction in gametocyte transcript prevalence in the subsequent month when compared to HIV-1 negative volunteers treated with AL. To assess gametocyte transmissibility, volunteer blood samples were used in standard membrane feeding assays (SFMA) with laboratory-reared A. gambiae, with evidence of transmission confirmed by at least one of 25 dissected mosquitoes per sample positive for at least one midgut oocyst. HIV-1 status, CD4+ T-cell levels and hematocrit were not significantly associated with successful transmission to A. gambiae. Analysis of SMFA blood samples revealed that 50% of transmission-positive blood samples failed to test positive by Plasmodium-specific 18S ribosomal RNA quantitative PCR (qPCR) and 35% failed to test positive for any gametocyte specific transcript marker by droplet digital (ddPCR), documenting that transmission occurred in the absence of molecular parasite/gametocyte detection. Overall, these findings highlight the complexity of HIV-1 malaria co-infection and the need to further define the unpredictable role of asymptomatic parasitemia in transmission to mosquitoes.</p

Polyclonal antibodies to PfEMP1 variants from laboratory strains show surface reactivity and rosette inhibition with <i>P. falciparum</i> clinical isolates.

<p>a) Clinical isolates were tested with PfEMP1 antibodies and controls for surface reactivity by live cell IFA (0.4 mg/ml) and rosette inhibition (1 mg/ml). The rosette frequency (RF), percentage of IgM-positive IEs (IgM+) and percentage of PfEMP1 antibody positive IEs (PfEMP1+, positive with either HB3var6 or TM284var1 antibodies) are shown for each isolate. The PfEMP1 antibodies that showed surface staining with each isolate are indicated by the shaded boxes. Positive surface staining was defined as punctate fluorescence specific to live IEs by IFA (as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g002" target="_blank">Figure 2</a>). The percentage rosette inhibition is shown inside each rectangle for all isolate/antibody combinations with >25% rosette inhibition. The controls are as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g004" target="_blank">Figure 4b</a>, and the Anti-Ros Pool is as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g005" target="_blank">Figure 5b</a>. The Anti-Ros pool was tested for rosette inhibition only. The dotted line separates isolates in which RF closely matches the percentage of IgM-positive IEs (above) from those in which the percentage of IgM-positive positive IEs is substantially lower than the rosette frequency (below). b) Flow cytometry of clinical isolate MAL43 with 0.4 mg/ml of total IgG from a non-immunised rabbit (negative control, left panel) and antibodies to TM284var1 (middle panel). IEs stained with Hoechst are in the right half, and antibody-positive IEs stained with Alexa Fluor 488 are in the upper right quadrant. An overlay of histograms (right panel) shows a clear population of stained IEs (blue line, second peak) distinct from the rabbit IgG control (red line, single peak). c) Five clinical isolates were tested by flow cytometry with the PfEMP1 antibody and control panel. The histograms show the negative controls, anti-PfEMP1 positive and IgM-positive IEs. The “negative PfEMP1 Ab” was antibody to TM180var1 and the IgM-negative control was a mouse IgG1 isotype control.</p

Polyclonal antibodies to PfEMP1 recognize the surface of homologous and heterologous live IEs.

<p>a) An example of the determination of the immunofluorescence end titre. Flow cytometry histograms showing the titration of antibodies to ITvar60 against IT/PAR+ parasites, compared to a non-immunized rabbit IgG control. The end titre (defined here as the lowest concentration of antibody giving surface staining above rabbit IgG background levels of more than 50% of the positive IE subpopulation) was 0.1 µg/ml. b) PfEMP1 antibodies (four-fold dilutions of total IgG starting at 400 µg/ml) were tested in IFA or flow cytometry against <i>P. falciparum</i> laboratory strains with various different adhesion phenotypes as indicated. The end titre for each antibody/parasite combination is shown inside each rectangle, with homologous antibody/parasite combinations being outlined in bold. Negative controls were non-immunized rabbit IgG control, and antibodies against NTS-DBLα from a non-rosetting Group A PfEMP1 variant (Non-ros Group A: HB3var3, expressed by HB3-HBEC which are non-rosetting parasites selected for binding to human brain endothelial cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Claessens2" target="_blank">[83]</a>). *The HB3R+ parasites contain a subpopulation of non-rosetting HB3var3-expressing IEs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665.s007" target="_blank">Table S1</a>) that are distinct from the IgM-positive HB3var6-expressing rosetting IEs.</p

Polyclonal antibodies to PfEMP1 inhibit rosetting and induce phagocytosis of heterologous rosetting laboratory strains.

<p>a) Rosette inhibition assays to determine the dose-dependent effects of PfEMP1 antibodies on homologous and heterologous rosetting laboratory strains. Data are compared to a control with no added antibody, which contained at least 40% of IEs in rosettes. Mean and standard deviation of triplicate values are shown. IC50: concentration of antibody giving 50% rosette inhibition. b) Rosette inhibition assay as above with 1 mg/ml of antibody, except for the Anti-Ros pool which consisted of a mixture of 0.1 mg/ml of each antibody (to HB3var6, TM284var1, ITvar60, Muz12var1, TM180var1 and ITvar9). Controls are as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g004" target="_blank">Figure 4b</a>. c) Phagocytosis assay of opsonised IT/PAR+ IEs co-incubated with the monocytic cell line Thp-1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Ghumra1" target="_blank">[13]</a>. Data are shown as percentage of the positive control opsonised with a rabbit anti-human erythrocyte antibody. Both homologous and heterologous antibodies induce phagocytosis of IT/PAR+ IEs. Control Rab: negative control of IgG from a non-immunized rabbit.</p