Characterization of novel malaria vaccine candidates representing alpha-helical coiled coil domains

The future vision in the battle against malaria goes beyond controlling the disease. Envisioned is the world-wide eradication of malaria. A substantial contribution to reach this goal is the development of an effective vaccine. Today’s most advanced and most effective malaria vaccine, RTS,S/AS01, showed efficacy of 30 to 66% against all clinical episodes. There is a great need to increase efficacy by the next generation malaria vaccines. A strategy for increasing RTS,S efficacy could be to combine it with an effective blood stage vaccine. The disappointing outcomes of clinical trials conducted for most current blood stage vaccines demands the identification of novel promising candidates. Under persistent exposure individuals develop immunity that protects against clinical disease but not parasitemia. This natural acquired immunity develops slowly and is reached in adolescence. In contrast, immunity against severe disease develops already after few infections. The mechanisms that underlie naturally acquired immunity or severe disease immunity remain poorly understood. Antibodies were demonstrated to play a critical role for controlling blood stage infection. It remains unclear which proteins elicit the production of protective antibodies and through which antibody effector function protection is provided. The relevance of antibodies in blood stage protection has the consequence that the immunogen correctly mimic the three-dimensional structure of the native protein. This PhD thesis has its major focus on immunogens that adopt a stable tertiary structure in aqueous environment. The availability of the P. falciparum genome sequences, transcriptome and proteome data has opened the avenue for the identification of novel targets for vaccine development. However, blood stage vaccine development has focused on only a few candidates. Previously our collaborators in this project have identified promising candidates by genome-wide screening for alpha-helical coiled coil domains in proteins expressed in the erythrocytic parasite stages. The segments with high probability score for coiled coil formation where selected. The 166 coiled coil segments derived from 131 proteins representing 4% of the blood stage proteome. 95 coiled coil fragments of a length of 30-40 amino acids were synthesized and analyzed systematically in a pre-clinical evaluation pathway. The aim of this thesis was to fill the gaps in the preclinical evaluation pathway of novel synthetic peptide vaccine candidates. The extensive polymorphism found in most parasite antigens represents a major obstacle for the development of efficacious blood stage vaccines. The genetic diversity of the identified coiled coil protein segments was studied in great detail. We found that coiled coil segments are well conserved, 82% of all selected 166 segments showed complete sequence conservation. Polymorphism was found predominantly in segments containing almost perfect tandem repeats. Based on these findings an optimized bioinformatic selection strategy was formulated proposing to exclude coiled coil segments consisting of almost perfect tandem repeats. The availability of basic knowledge about vaccine candidates is a prerequisite for vaccine development and is essential to attract further funding for continued clinical development. A detailed cell biological characterization was undertaken for the most promising candidate, PFF0165c (newly termed Trophozoite exported protein 1 (Tex1)) Transcript and protein levels were analyzed throughout the intra-erytrocytic development cycle. Tex1 transcripts were found up-regulated in the early trophozoite stage. This was supported by Tex1 protein levels. Tex1 abundance persisted until parasite egress. Immunofluorescence experiments revealed that Tex1 is exported and associates to parasite-derived structures, termed Maurer’s clefts. Before parasite egress Tex1 resides in close proximity to the red blood cell membrane. In the search of sequence motifs responsible for Tex1 export we found that the actual translational start site is positioned 43 amino acids upstream of the start site previously predicted. The additional 43 amino acids function as signal peptide, directing the protein into the classical secretory pathway. This thesis contributed to the immunological characterization of the intrinsically unstructured region (P27A) of Tex1. P27A was evaluated for vaccine potential and met the principal requirements to be downselected for a phase 1 trial. P27A was recognized by a majority of naturally exposed individuals, highly immunogenic, highly conserved and P27A-specific human and mouse sera were effective in in vitro parasite killing by Antibody-dependent cellular inhibition assay. High P27A-specifc antibody titers were found to positively correlate with protection. Clinical grade P27A peptide is currently produced. In order to validate synthetic peptides as antigens the recognition by sera of adults from endemic region was compared to the recognition of the antigen recombinant expressed in E. coli. Comparable recognition of both types of antigens was observed. This thesis provides evidence that the approach initiated by our collaborators is invaluable. This strategy, if proven successful in clinical trials, could be applied for vaccine development against many other pathogens from which genome data is available

Predicting Major Adverse Events in Patients With Acute Myocardial Infarction

Early and accurate detection of short-term major adverse cardiac events (MACE) in patients with suspected acute myocardial infarction (AMI) is an unmet clinical need.; The goal of this study was to test the hypothesis that adding clinical judgment and electrocardiogram findings to the European Society of Cardiology (ESC) high-sensitivity cardiac troponin (hs-cTn) measurement at presentation and after 1 h (ESC hs-cTn 0/1 h algorithm) would further improve its performance to predict MACE.; Patients presenting to an emergency department with suspected AMI were enrolled in a prospective, multicenter diagnostic study. The primary endpoint was MACE, including all-cause death, cardiac arrest, AMI, cardiogenic shock, sustained ventricular arrhythmia, and high-grade atrioventricular block within 30 days including index events. The secondary endpoint was MACE + unstable angina (UA) receiving early (≤24 h) revascularization.; Among 3,123 patients, the ESC hs-cTnT 0/1 h algorithm triaged significantly more patients toward rule-out compared with the extended algorithm (60%; 95% CI: 59% to 62% vs. 45%; 95% CI: 43% to 46%; p < 0.001), while maintaining similar 30-day MACE rates (0.6%; 95% CI: 0.3% to 1.1% vs. 0.4%; 95% CI: 0.1% to 0.9%; p = 0.429), resulting in a similar negative predictive value (99.4%; 95% CI: 98.9% to 99.6% vs. 99.6%; 95% CI: 99.2% to 99.8%; p = 0.097). The ESC hs-cTnT 0/1 h algorithm ruled-in fewer patients (16%; 95% CI: 14.9% to 17.5% vs. 26%; 95% CI: 24.2% to 27.2%; p < 0.001) compared with the extended algorithm, albeit with a higher positive predictive value (76.6%; 95% CI: 72.8% to 80.1% vs. 59%; 95% CI: 55.5% to 62.3%; p < 0.001). For 30-day MACE + UA, the ESC hs-cTnT 0/1 h algorithm had a higher positive predictive value for rule-in, whereas the extended algorithm had a higher negative predictive value for the rule-out. Similar findings emerged when using hs-cTnI.; The ESC hs-cTn 0/1 h algorithm better balanced efficacy and safety in the prediction of MACE, whereas the extended algorithm is the preferred option for the rule-out of 30-day MACE + UA. (Advantageous Predictors of Acute Coronary Syndromes Evaluation [APACE]; NCT00470587)

Sequence Conservation in Plasmodium falciparum α-Helical Coiled Coil Domains Proposed for Vaccine Development

BACKGROUND: The availability of the P. falciparum genome has led to novel ways to identify potential vaccine candidates. A new approach for antigen discovery based on the bioinformatic selection of heptad repeat motifs corresponding to alpha-helical coiled coil structures yielded promising results. To elucidate the question about the relationship between the coiled coil motifs and their sequence conservation, we have assessed the extent of polymorphism in putative alpha-helical coiled coil domains in culture strains, in natural populations and in the single nucleotide polymorphism data available at PlasmoDB. METHODOLOGY/PRINCIPAL FINDINGS: 14 alpha-helical coiled coil domains were selected based on preclinical experimental evaluation. They were tested by PCR amplification and sequencing of different P. falciparum culture strains and field isolates. We found that only 3 out of 14 alpha-helical coiled coils showed point mutations and/or length polymorphisms. Based on promising immunological results 5 of these peptides were selected for further analysis. Direct sequencing of field samples from Papua New Guinea and Tanzania showed that 3 out of these 5 peptides were completely conserved. An in silico analysis of polymorphism was performed for all 166 putative alpha-helical coiled coil domains originally identified in the P. falciparum genome. We found that 82% (137/166) of these peptides were conserved, and for one peptide only the detected SNPs decreased substantially the probability score for alpha-helical coiled coil formation. More SNPs were found in arrays of almost perfect tandem repeats. In summary, the coiled coil structure prediction was rarely modified by SNPs. The analysis revealed a number of peptides with strictly conserved alpha-helical coiled coil motifs. CONCLUSION/SIGNIFICANCE: We conclude that the selection of alpha-helical coiled coil structural motifs is a valuable approach to identify potential vaccine targets showing a high degree of conservation

Predisposing factors for Hoffa’s fat pad syndrome: a systematic review

Abstract Background Hoffa’s fat pad syndrome has been defined as impingement of Hoffa’s fat pad, leading to oedema and fibrosis. The primary aim of this systematic review was to identify morphological differences in Hoffa’s fat pad between patients with and without Hoffa’s fat pad syndrome, evaluating them as risk factors predisposing to its development. The secondary aim was to summarize and evaluate current evidence pertaining to the management of Hoffa’s fat pad syndrome. Materials and Methods The protocol for this review was prospectively registered (PROSPERO registration: CRD42022357036). Electronic databases, currently registered studies, conference proceedings and the reference lists of included studies were searched. All studies evaluating differences in Hoffa’s fat pad anatomy under imaging between patients with and without Hoffa’s fat pad syndrome were included, as well as those exploring epidemiological factors predisposing to its development (ethnicity, employment status, sex, age and BMI), and studies reporting on the effect of treatment on Hoffa’s fat pad morphology. Results A total of 3871 records were screened. Twenty one articles satisfied the inclusion criteria, evaluating 3603 knees of 3518 patients. Patella alta, increased tibial tubercle-tibial groove distance, and increased trochlear angle were found to predispose the development of Hoffa’s fat pad syndrome. Trochlear inclination, sulcus angle, patient age and BMI were not associated with this condition. The link between Hoffa’s fat pad syndrome and ethnicity, employment, patellar alignment, Hoffa’s fat pad composition, physical activity and other pathological processes cannot be established due to lack of evidence. No studies reporting on treatment for Hoffa’s fat pad syndrome were identified. Though weight loss and gene therapy may provide symptomatic relief, further research is required to corroborate these claims. Conclusion Current evidence suggests that high patellar height, TT-TG distance, and trochlear angle predispose the development of Hoffa’s fat pad syndrome. In addition, trochlear inclination, sulcus angle, patient age and BMI do not seem to be associated with this condition. Further research should explore the link between Hoffa’s fat pad syndrome and sport as well as other conditions pertaining to the knee. In addition, further study evaluating treatment approaches for Hoffa’s fat pad syndrome is required

Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1). In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal

Brefeldin A sensitivity of Tex1 export.

<p>3D7 infected RBC were treated with BFA and fixed (+BFA). Tex1 was stained using P27 or P27A-specific mouse antibodies (in red, upper panel: early trophozoite, middle panel: trophozoite). Tex1 visible inside the parasite in close proximity to the nucleus. A control culture (+ETOH) was incubated with equivalent concentration of ethanol, the solvent of Brefeldin A. In the control culture Tex1 was correctly exported and associated to MC (in red). The nucleus was stained with DAPI (in blue). Transmission image (DIC). scale bar: 5 µm.</p

Immunofluorescence staining of erythrocytes infected by <i>P. falciparum</i> (ring, trophozoites and schizont stages) using P27-specific polyclonal rabbit sera.

<p>P27-specific polyclonal rabbit sera was used to detect Tex1 (green) A) in late ring stages B) in trophozoite stages C) in schizont stages. Nucleus stained with DAPI (blue), transmission picture of the infected red blood cell (DIC) and merged picture of the two signals or the signals merged with transmission picture (merge), Scale bar: 5 µm.</p

Equinatoxin II assay.

<p>A) 3D7 infected RBC lysed with equinatoxin II. Integrity of MCs is demonstrated by the absence of the SBP1 signal after using SBP1 N-terminus specific polyclonal mouse sera (note: N-terminus of SBP1 faces the lumen of MCs). Tex1 signal on the MC surface was obtained with P27-specific polyclonal rabbit sera (in green). B) 3D7 infected RBC lysed with equinatoxin followed by Triton lysis. MC lumen is now accessible for antibodies as shown by the SBP1 signal (in red). Nuclear DNA stained with DAPI (blue), Transmission image (DIC). Scale bar: 5 µm.</p