Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230

Pfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A “functional” antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443–1274 range, and all contained aa 543–730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918–1274 region. Within aa 443–917, further analysis showed the existence of functional epitopes not only within the aa 543–730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543–588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02

Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

Determination of Specific IgG to Identify Possible Food Intolerance in Athletes Using ELISA

Nutrition is considered one of the foundations of athletic performance, and post-workout nutritional recommendations are fundamental to the effectiveness of the recovery and adaptive processes. Therefore, at present, new directions in dietetics are being formed, focused on the creation of personalized diets. To identify the probable risk of somatic and allergic reactions upon contact with food antigens, we used the method of enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of IgG antibodies in the blood plasma of athletes against protein–peptide antigens accommodated in food. The study enrolled 40 athletes of boating and fighting sport disciplines. We found that the majority of the studied participants were characterized by an elevated IgG level against one or two food allergens (barley, almond, strawberry, etc.). Comparative analysis of the semiquantitative levels of IgG antibodies in athletes engaged in boating and fighting did not reveal significant differences between these groups. As a result, foods that are likely to cause the most pronounced immune response amongst the studied participants can be identified, which may indicate the presence of food intolerances. An athlete’s diet is influenced by both external and internal factors that can reduce or worsen the symptoms of a food intolerance/allergy associated with exercise. The range of foods is wide, and the effectiveness of a diet depends on the time, the place, and environmental factors. Therefore, during the recovery period (the post-competition period), athletes are advised to follow the instructions of doctors and nutritionists. An effective, comprehensive recovery strategy during the recovery period may enhance the adaptive response to fatigue, improving muscle function and increasing exercise tolerance. The data obtained may be useful for guiding the development of a new personalized approach and dietary recommendations covering the composition of athletes’ diet and the prevalence of food intolerance.

The synthesis and biological evaluation of glycosphingolipids for improved cancer immunotherapy

<p>The immune system plays a crucial role in providing the first line of defence against invading pathogens such as bacteria, viruses and parasites. It is activated when immune cells known as dendritic cells (DCs) detect specific molecules that are foreign to the host, and present them to T cells. This in turn causes the activation of T cells, which marks the start of an immune response leading to the clearance of the invader. The pathogen-derived molecules recognised by the immune cells are typically peptides and their role as activators of the immune system is well established. While T cells were originally thought to only recognise peptide antigens, it is now evident that T cells are also able to recognise nonpeptide antigens. Recognition of non-peptide antigens confers protection against pathogens that have cell surfaces that are highly functionalised with carbohydrate moieties, such as glycolipids, glycopeptides and polysaccharides. Specifically, glycosphingolipids (GSLs) can activate invariant Natural Killer T (iNKT) cells via their T cell receptor (TCR) when presented by the CD1d molecule found on the surface of DCs. α-Galactosylceramide (α-GalCer 1, Figure 1), a synthetic analogue of a GSL extracted from the marine sponge Agelas mauritianus, was discovered to be a potent stimulator of iNKT cells when presented by CD1d. α-GalCer is currently being used in clinical trials as an adjuvant to boost the activation of immune cells during cancer immunotherapy. Although the molecular interaction of α-GalCer with CD1d and iNKT cells is well established, it is not fully understood how the glycolipid interacts with different subsets of DCs. Greater understanding of the fate of the glycolipid during cancer immunotherapy will provide crucial information on how the current therapy can be improved. In this thesis, the design and synthesis of two fluorescent α-GalCer probes, dansyl-α-GalCer (2, Figure 1) and BODIPY-α-GalCer (3, Figure 1) is reported. Dansyl-α-GalCer was able to activate DCs and iNKT cells in a similar fashion to the parent glycolipid α-GalCer. Its activity was CD1d-dependent and DCs that have taken up α-GalCer in vitro can be detected by flow cytometry. Unfortunately, the fluorescence of dansyl-α-GalCer was too weak to be detected by fluorescent microscopy due to photobleaching of the dye. Accordingly, another α-GalCer probe bearing a brighter fluorescent group, BODIPY, was synthesised. The α-GalCer probes were made via two synthetic strategies and the benefits and shortcomings of each synthetic route are discussed. Isoglobotrihexosylceramide (4, iGb3, Figure 2) is another GSL known to activate iNKT cells. Like α-GalCer, it is presented by DCs in the context of a CD1d molecule. iGb3 contains a sphingosine lipid backbone β-linked to a trisaccharide head group, which is in contrast to the α-linked phytosphingosine lipid found on α-GalCer. Despite the structural difference, iGb3 can stimulate iNKT cells, though to a lesser extent than α-GalCer. The intriguing activity of iGb3 provides a platform to further investigate the molecular interactions between CD1d, glycolipid and TCR of iNKT cell. The crystal structure of iGb3 in complex with mouse CD1d and TCR of mouse iNKT cell show compelling evidence that the terminal galactose moiety is crucial for the observed activity and this is attributed to the hydrogen bond between the 6´´´-OH and Thr159 on the CD1d. To unambiguously determine the importance of the hydrogen bond conferred by 6´´´-OH, 6´´´-deoxy-iGb3-sphingosine (5, Figure 2) was synthesised. 6´´´-deoxy-iGb3-sphinganine 6 was also synthesised to study the role of the double bond on the sphingosine backbone. A novel synthetic route for the synthesis of iGb3 analogues was established. This allowed for the expedient synthesis of 6´´´-deoxy-iGb3 derivatives that will subsequently be crystallised with CD1d and TCR of iNKT cell, to provide further insight into the structural requirements of β-linked GSLs. Studies have also revealed that the length and saturation of the N-acyl chain of GSLs greatly influences their activity. It is speculated that varying the length of the acyl chain affects the processing and loading of the glycolipid onto CD1d and also TCR binding affinity. To this end, the syntheses of a series of acyl chain analogues of iGb3, including the shorter chain homologue C12:0 7 (Figure 3) and the unsaturated C20:2 derivative 8 are reported. A divergent synthetic route was employed, whereby a common intermediate from the synthesis of 6´´´-deoxyiGb3 was used. This allowed for efficient syntheses of the acyl chain analogues that will facilitate a greater understanding of the structure-activity relationships. Taken together, the GSLs synthesised provide crucial insight into how they modulate the immune system and will guide future optimisation of cancer immunotherapy regimes.</p>

Tumor Antigen-Based Nanovaccines for Cancer Immunotherapy: A Review

As an important means of tumor immunotherapy, tumor vaccines have achieved exciting results in the past few decades. However, there are still many obstacles that hinder tumor vaccines from achieving maximum efficacy, including lack of tumor antigens, low antigen immunogenicity and poor delivery efficiency. To overcome these challenges, researchers have developed and investigated various new types of tumor antigens with higher antigenic specificity and broader antigen spectrum, such as tumor-specific peptide antigens, tumor lysates, tumor cell membrane, tumor associated exosomes, etc. At the same time, different nanoparticulate delivery platforms have been developed to increase the immunogenicity of the tumor antigens, for example by increasing their targeting efficiency of antigen-presenting cells and lymph nodes, and by co-delivering antigens with adjuvants. In this review, we summarized different types of the tumor antigens that have been reported, and introduced several nanovaccine strategies for increasing the immunogenicity of tumor antigens. The review of recent progress in these fields may provide reference for the follow-up studies of tumor antigen-based cancer immunotherapy.

A Systematic Interrogation of MHC Class I Antigen Presentation Identifies Constitutive and Compensatory Protein Degradation Pathways

Protein degradation products are constitutively presented as peptide antigens by MHC Class I. While hypervariability of Class I genes is known to tremendously impact antigen presentation, whether differential function of protein degradation pathways (comprising >1000 genes) could alter antigen generation remains poorly understood apart from a few model substrates. In this study, we introduce normalization methods for quantitative antigen mass spectrometry and confirm that most Class I antigens are dependent on ubiquitination and proteasomal degradation. Remarkably, many antigens derived from mitochondrial inner membrane proteins are not. Additionally, we find that atypical antigens can arise from compensatory protein degradation pathways, such as an increase in mitochondrial and membrane protein antigen presentation upon proteasome inhibition. Notably, incomplete inhibition of protein degradation pathways may have clinical utility in cancer immunotherapy, as evidenced by appearance of novel antigens upon partial proteasome inhibition.

Monoclonal antibodies targeting surface exposed epitopes of Candida albicans cell wall proteins confer in vivo protection in an infection model

MAb based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development with currently no licensed antifungal mAbs available. The cell wall glycoproteins of Candida albicans are potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. We describe phage display based generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans - Utr2 and Pga31, using peptide antigens representing the surface exposed regions of CWPs at elevated levels during in vivo infection. Reformatted mAbs preferentially recognised C. albicans hyphal forms compared to yeast cells and an increased binding in cells pre-treated with caspofungin. In macrophage interaction assays, mAb pre-treatment resulted in a faster engulfment of C. albicans cells suggesting opsonophagocytosis. Finally, in a series of clinically predictive, mouse models of systemic candidiasis, our lead mAb achieved an improved survival (83%) and several log reduction of fungal burden in the kidneys, similar to levels achieved for the fungicidal drug caspofungin, and superior to any anti-Candida mAb.

Optimizing use of multi-antibody assays for Lyme disease diagnosis: A bioinformatic approach

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.

Abstract MP53: A Role Of Anti-isolevuglandin-adduct Antibody Production In Hypertension

Isolevuglandins (isoLG) are products of lipid oxidation that covalently ligate self-proteins and contribute to immune activation in hypertension. We hypothesized that these are recognized as “non-self” elicit an antibody response. To determine the presence of anti-isoLG adduct antibodies in humans, immunoblots were performed using endogenous antibodies present in plasma of healthy and hypertensive subjects against human proteins artificially adducted with isoLG. Protein-G HRP was used to visualize the binding of IgG antibodies. We found that humans with hypertension possess IgG antibodies that bind isoLG protein adducts to a greater extent compared to unadducted protein. To identify unique monoclonal antibodies to isoLG adducts C57Bl/6 mice were made hypertensive via angiotensin II infusion for two weeks and then boosted with kidney protein that was adducted with isoLG. Boosted mice were then sacrificed and splenic B-cells were fused with a myeloma cell line. Individual colonies were screened for reactivity with isoLG adducts identifying 11 monoclonal antibody clones with a high specificity for isoLG adducts. Four clones were chosen for further analysis that exhibited 1.5 to 6-fold affinity to adducted vs unadducted mouse proteins. Western blots of normotensive and hypertensive kidney, bone marrow, and heart protein lysate using one of our monoclonal antibodies (clone 2B11) revealed a 90 kDa protein that is increased in bone marrow of hypertensive mice. We conclude that hypertension results in the production of anti-isoLG adduct antibodies in mice and humans. Identification of unique monoclonal antibodies that react with isoLG adducts suggests clonal expansion of anti-isoLG antibody producing cells. Finally, a hypertensive antigen is recognized by the 2B11 clone. Future studies will utilize these antibodies to identify peptide antigens that drive immune activation in hypertension.