Evaluation of candidate peptides for the immunisation against angiotensin II

Arterielle Hypertonie betrifft bis zu 600 Millionen Menschen weltweit. Chronisch erhöhter Blutdruck ist ein großer Risikofaktor für Herz-Kreislauferkrankungen. Das sogenannte Renin-Angiotensin System (RAS) erhält das Gleichgewicht zwischen gefäßverengenden und gefäßerweiternden Effekten auf den Blutkreislauf. Ein wichtiger Bestandteil dieses Systems ist Angiotensin II (Ang II). Dieses aus acht Aminosäuren zusammengesetzte Peptid wirkt gefäßverengend und induziert die Wiederaufnahme von Wasser aus dem Urin ins Blut. Dadurch erhöht Ang II den Blutdruck. Angiotensin (1-7) (Ang 1-7), ein weiteres RAS- Peptid, hingegen hat eine gegensätzliche Wirkung auf das Herz-Kreislaufsystem. Verschiedene niedermolekulare Substanzen werden eingesetzt, um die Produktion oder die Wirkung von Ang II zu blockieren und somit den Blutdruck zu senken. Diese müssen täglich eingenommen werden und zeigen nur in Kombination Effektivität. Daher ist es ein wichtiges Anliegen, neue effektive Therapien gegen Bluthochdruck und seine fatalen Folgen hervorzubringen. Die Entwicklung eines Impfstoffs gegen Ang II könnte hier einen wesentlichen Beitrag leisten. Die vorliegende Arbeit ist Teil eines Projekts, das darauf abzielt einen Peptid-Impfstoff zu entwickeln, der es ermöglicht Ang II aus dem System zu entfernen, ohne Ang 1-7 oder seinen Vorläufer, Angiotensin I (Ang I), anzutasten. Hierfür wurde die Immunogenität verschiedener Ang II-Peptid Varianten getestet, sowie deren Fähigkeit überprüft, Seren mit unterschiedlicher Reaktivität zu induzieren. Daten aus verschiedenen ELISA (enzyme linked immunosorbent assay)- Experimenten zeigen, dass Peptid- Varianten, auch Variotope® genannt, selektiert werden konnten, die Antikörper induzieren, welche eine stark reduzierte Reaktivität gegen Ang 1-7 und Ang I aufweisen, das Ang II Peptid jedoch nach wie vor effektiv binden. Ein zellulärer Assay soll weiters die Überprüfung der inhibitorischen Fähigkeit von Variotop®-induzierten Seren in vitro ermöglichen. Der hier entwickelte Assay basiert auf der Expression von Luziferase durch die Aktivierung von NF-κB in HEK293 Zellen. Stimulation der, zusätzlich mit einem Angiotensin II Rezeptor-GFP Konstrukt transient transfektierten, HEK293 Zellen mit Ang II bewirkt einen signifikanten Signal-Anstieg. Die große Differenz zwischen unstimuliertem und stimuliertem Zustand, ermöglicht die Bestimmung der blockierenden Wirkung von Ang II- Antikörpern.Hypertension is the most prevalent risk factor for cardiovascular disease (CVD) and thus constitutes an important worldwide public-health challenge. The renin-angiotensin system (RAS) maintains the balance of contractive and dilutive effects within the vasculature. Angiotensin II (Ang II), an octapeptide, is a potent mediator of vasoconstriction and anti-diuretic effects and thus elevates blood pressure. Angiotensin (1-7) (Ang 1-7), a heptapeptide derived from angiotensin I (Ang I) or Ang II via angiotensin-converting enzyme (ACE) or angiotensin-converting enzyme II (ACE2) respectively is known to counteract these actions. Vaccination therapies targeting a component of the RAS other than Ang II have so far been unsuccessful in achieving target blood pressure or have even induced autoimmune disease. However, a phase II clinical study using Ang II as a target for immunotherapy conducted has been successful in decreasing blood pressure levels in humans. AFFiRiS AG is developing a peptide-based hypertension vaccine targeting Ang II, while sparing Ang I and Ang 1-7 to maintain its vasodilative potential. Therefore, different candidates of Ang II-derived peptide-variants (Variotopes®) are evaluated for immunogenicity and specificity for Ang II. ELISA results show that it is possible to induce higher antibody titres against Ang II (a measurement for immunogenicity) while reducing the crossreactivity of antibodies to Ang I and Ang 1-7 with Variotopes®. The ability of Variotope®-induced antibodies to block the binding of Ang II to its receptor should be determined with a functional assay based on cell cultures. A luciferase activity assay has been established to measure the effects of Ang II stimulation in HEK293 cells transiently co-transfected with AT1R-EGFP and NFkB-luciferase expression vectors. The luciferase activity is significantly higher in stimulated than in un-stimulated cells, thus giving enough margin to determine inhibitory properties of antibodies. Furthermore, this signal increase could be reversed with Angiotensin II type I receptor blocker losartan. The established assay can be used to determine the potential correlation between antibody titre, affinity and functionality

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis

Immunization with apical membrane antigen 1 confers sterile infection-blocking immunity against Plasmodium sporozoite challenge in a rodent model

Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8 and CD4 T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity

Les Travaux sur le champ littéraire. Enjeux, acquis, perspectives

Infectious diseases remain a leading global cause of morbidity and mortality and there is an urgent need for effective approaches to develop vaccines, especially against complex pathogens. The availability of comprehensive genomic, proteomic and transcriptomic datasets has shifted the paradigm of vaccine development from microbiological to sequence-based approaches. However, how to effectively translate raw data into candidate vaccines is not yet obvious. Herein, we review cutting-edge technologies and screening strategies to mine genomic sequence information for state-of-the-art rational vaccine design, and highlight recent trends. Interdisciplinary approaches which cross the traditional boundaries of genomics, molecular biology, cell biology, immunology and computer science, and which prioritise antigens according to clinically relevant criteria, offer potential solutions to the widespread threat that complex pathogens pose to public health

Novel Plasmodium antigens identified via genome-based antibody screen induce protection associated with polyfunctional T cell responses

The development of vaccines against complex intracellular pathogens, such as Plasmodium spp., where protection is likely mediated by cellular immune responses, has proven elusive. The availability of whole genome, proteome and transcriptome data has the potential to advance rational vaccine development but yet there are no licensed vaccines against malaria based on antigens identified from genomic data. Here, we show that the Plasmodium yoelii orthologs of four Plasmodium falciparum proteins identified by an antibody-based genome-wide screening strategy induce a high degree of sterile infection-blocking protection against sporozoite challenge in a stringent rodent malaria model. Protection increased in multi-antigen formulations. Importantly, protection was highly correlated with the induction of multifunctional triple-positive T cells expressing high amounts of IFN-γ, IL-2 and TNF. These data demonstrate that antigens identified by serological screening are targets of multifunctional cellular immune responses that correlate with protection. Our results provide experimental validation for the concept of rational vaccine design from genomic sequence data

Accuracy of parasite cDNA detection.

<p>The standard deviation for four replicates of one sample was divided by the mean value for the target gene and plotted against the log of the mean parasite-derived cDNA copies/reaction. Each point represents the mean of four replicates of one mouse liver cDNA sample. A cut-off value of 8x10^-3 for the ratio between standard deviation and mean is suggested for accurate quantitation. The dotted line marks the mean value between the highest amount of parasite cDNA measured with a standard deviation/mean ratio below 8x10^-3 and the highest amount of parasite cDNA measured with a standard deviation/mean ratio above 8x10^-3, for (A) Py18S qRT-PCR (1.6 copies/reaction) and for (B) PyCytB qRT-PCR (66.5 copies/reaction).</p

Recovery rate.

<p>A cDNA sample extracted from the liver of a naïve mouse was spiked with 7x10⁵, 7x10³ or 70 ‘plasmid equivalents’ of Py18S control plasmid. C_q values of spiked samples were plotted on the y-axis against the given copy number of plasmid. Linear regression analysis reveals a robust linear correlation of calculated to given copy numbers (R²=0.99). Data are presented as mean values of three technical replicates; error bars represent the standard deviation.</p

Highly sensitive quantitative real-time PCR for the detection of Plasmodium liver-stage parasite burden following low-dose sporozoite challenge

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy