Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents. / QC 20111117 / Development and applications of protein microarrays / The Swedish Human Proteome Resource (HPR) program

Bacteria are coated with repeating units of oligosaccharides that exhibit remarkable diversity. Often, glycan units of three or even two sugars are sufficient to identify a species of bacteria. Such specificity makes bacterial surface glycans attractive vaccine targets. However, efforts to create effective vaccines against carbohydrates have been hampered by poor vaccine design as well as the human immune tendency to respond to glycan antigens with non-specific, T-cell independent mechanisms. As a result, carbohydrate vaccines have historically produced only adequate memory responses in healthy individuals and poor responses in the elderly or immunocompromised. To circumvent these issues, a novel conjugate vaccine was developed that utilizes theQβ virus-like particle carrier that displays both a carbohydrate antigen as well as a Natural Killer T cell adjuvant. This unique vaccine has been reported to stimulate the production of high affinity (nanomolar) antibodies against carbohydrate antigens. To further conjugate vaccine research, the present work synthesizes two bacterial surface antigens: a trisaccharide from Streptococcus pneumoniae serotype 23F (Sp23F), and a pentasaccharide from Ruminococcus gnavus (Rg). Sp23F has been characterized as one of the more virulent and disease-causing strains of S. pneumoniae. Rg secretes highly immunostimulatory proteins and is associated with irritable bowel syndrome. The Sp23F antigen is synthesized with an alkyne at the reducing end of the sugar to facilitate coupling to Qβ. A selection reagent for Sp23F is also synthesized to enable the extraction of antibodies and B cells that bind the antigen. In conjunction with providing a conjugate vaccine antigen, the Rg pentasaccharide will be examined as a TLR4 ligand and was therefore synthesized without an alkyne. The Rg conjugate vaccine shows promise in treating irritable bowel syndrome as well as facilitating research into the role Rg plays in the human microbiome.

No description available.

The Shigella species are Gram-negative enteropathogens that produce severe diarrhea, cramping, and dehydration in millions of people annually. The pathogens most commonly infect children under the age of 5 years in developing nations, where the rise of multidrug-resistant species is increasingly problematic. Despite several attempts to develop a vaccine against these pathogens, no successful vaccine has been produced. In order to achieve this goal, several characteristics of Shigella must be further elucidated. Namely, we must better understand the mechanisms Shigella employs in order to circumvent the immune response. A key way in which Shigella circumvents the innate defenses of the host is through resistance to bile salts, the principal component of bile, a substance found in the small intestine that is required for digestion. One such bile salt resistance mechanism of Shigella involves lipopolysaccharide (LPS), an extracellular structure composed of three regions: a transmembrane lipid, a polysaccharide core, and an O-antigen. LPS and LPS modifications have been implicated in bile salts resistance in other enteropathogens. Thus, the goal of this study was to build from preliminary findings to understand the role of LPS in conferring bile salts resistance in Shigella. Two Shigella flexneri mutants were studied to understand the roles of the polysaccharide core and O-antigen on bacterial growth and LPS modifications during exposure to bile salts. Growth comparisons of the mutants relative to wild type bacteria in the presence of bile salts were performed, including analysis of growth with exposure to bile salts and with varying levels of environmental glucose. Additionally, LPS was extracted from wild type and mutant bacteria grown in these conditions for analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The growth curves demonstrated that both the O-antigen and polysaccharide core mutants exhibited slow growth with exposure to bile salts, while the SDS-PAGE analyses revealed changes in the LPS profile of wild type and both LPS mutants when grown in bile salts. These data indicate that the O-antigen likely has an important role in conferring bile salts resistance and that the polysaccharide core may also facilitate resistance. This study allows us to better understand how LPS contributes to bile salts resistance in S. flexneri, which may enhance efforts to develop an effective vaccine against this pathogen. / 2023-12-10T00:00:00Z

Multi-Antigen-Komplexe wurden in der Vakzinologie als effizientes Modell genutzt, um eine breite Impfstoffabdeckung gegen mehrere Stämme desselben Pathogens zu erzielen. Hier werden die Ergebnisse zur menschlichen B-Zell-Reaktion auf einen Multi-Antigen-Komplex (Bexsero) in drei Impfstoffen (Vax1, Vax2 und Vax3) dargestellt. Bexsero ist ein Impfstoff, der aus vier Antigenen (fHbp-GNA2091, NHBA-GNA1030, NadA und OMV (NZ98-254)) für Neisseria meningitidis (Nm) B besteht. Bei allen drei Impfstoffen konnten außerordentlich diverse Immunglobuline (Ig) beobachtet werden, die als Reaktion auf Bexsero mit einzigartigen Ig-Genselektionsmustern erzeugt wurden. Die Daten zeigen auch Igs, die eine Reihe von Spezifitäten aufweisen (Bexsero-spezifisch-reaktive Igs (nur Vax3) oder polyreaktive Igs (Vax2, Vax3 und Vax4)) und Affinitäten (hochbindende, mäßig bindende, schwach bindende und nicht reaktive Igs). Es wurde keine eindeutige Korrelation zwischen spezifischen Ig-Genmerkmalen und Ig-Reaktivitätseigenschaften beobachtet, obwohl Igs von allen Impfstoffen kollektiv unterschiedliche Affinitäten innerhalb/zwischen Cluster-Igs und zwischen Nicht-Clustern von Bexsero aufweisen, was potenzielle Vorteile für einen breiten Schutz mit sich bringt. Ig-Gen-Merkmale und Antigen-Reaktivitätseigenschaften von Igs, die gegen NHBA (22 Igs), fHbp (2 Igs) und NadA (2 Igs) erzeugt wurden, sind ebenfalls gezeigt. Diese Ig zeigten schwache Bindungsaffinitäten, wenn sie an endogen exprimierten Antigenen auf Nm mc58 getestet wurden, möglicherweise aufgrund eines ungeordneten N-Terminus von NHBA. Es wurde eine Anreicherung von hochmutierten polyreaktiven Ig beobachtet. Es werden unterschiedliche Immunoselektivitätsgrade für die verschiedenen Antigene beobachtet, was auf eine Antigenimmunodominanz sowie auf Hinweise auf eine Epitopmaskierung hindeutet. Mit einem kontrollierbaren System von 4 Antigenen eröffnen die Daten die Möglichkeit die menschliche B-Zell-Reaktion auf Multi-Antigen-Komplexe zu verstehen und zeigen, dass ein umfassendes Verständnis über die feinen zellulären und humoralen Einzelheiten der Immunantworten des Impfstoffs während klinischer Studien erforderlich ist. / Multi-antigen complexes have been exploited in vaccinology as an efficient model, to achieve broad vaccine coverage against multiple strains of the same pathogen. Here, the findings on the human B cell response to a multi-antigen complex (Bexsero) in three vaccinees (Vax1, Vax2 and Vax3) are shown. Bexsero is a vaccine comprising of four antigens (fHbp-GNA2091, NHBA-GNA1030, NadA and OMV (NZ98-254)) for Neisseria meningitidis (Nm) B. Immensely diverse (isotype distribution, IgVH and IgJH gene usage, CDR3 length distribution and clonal selection) immunoglobulins (Igs) generated in response to Bexsero with unique Ig gene selection patterns in all three vaccinees was observed. The data also shows Igs that exhibit a range of specificities {Bexsero-specific-reactive Igs (Vax3 Only) or polyreactive Igs (Vax2, Vax3 and Vax4)} and affinities (highly binding, moderately binding, weakly binding and unreactive Igs). No unique correlation between specific Ig gene features and Ig reactivity properties was observed, albeit Igs from all vaccinees collectively exhibit varied affinities within/between cluster Igs, and amongst non-clusters to Bexsero, with potential advantages for broad protection. Ig gene features and antigen-reactivity properties of Igs generated against NHBA (22 Igs), fHbp (2 Igs) and NadA (2 Igs) are also shown. These Igs exhibited weak binding affinities when tested on endogenously expressed antigens on Nm mc58, potentially due to disordered N-terminal of NHBA. Enrichment of highly mutated polyreactive Igs was observed. Varying degrees of immunoselectivity to the different antigens, suggesting antigen immunodominance as well as evidence of epitope masking are observed. With a controllable system of 4 antigens, the data opens a potential window to understanding the human B cell response to multi-antigen complexes and evinces the need for expansive understanding of the fine cellular and humoral details of vaccine immune responses during clinical trials.

In the normal ectocervix, mitoses are rare and are usually confined to the basal layers. In contrast, they occur more frequently in cervical intraepithelial neoplasia (CIN) and are seen at higher levels, suggesting that CIN may be associated with a progressive dysfunction in proliferative activity of cervical cells. The objective of this study was to use proliferating cell nuclear antigen (PCNA) immunohistochemistry to examine the proliferative activity of cervical epithelial cells in CIN lesions. Sixty- eight cervical biopsies were examined; 20 were totally benign, 14 had CIN I, 21 CIN II, and 13 CIN III. In benign epithelia, PCNA staining was usually confined to the basal layers, whereas in CIN the staining was seen at progressively higher levels of the epithelium. There was a statistically significant correlation between the CIN grade and the highest level of PCNA staining (PCNA grade, r = 0.746, P < 0.001). In addition, the PCNA grade showed significant correlation with the highest level at which mitoses were seen (mitosis grade, r = 0.706, P < 0.001), and a strong direct correlation between the mitosis and CIN grades was also observed (r = 0.955, P < 0.001). These data demonstrate that (1) PCNA immunoreactivity in neoplastic cervical epithelium is different from that seen in the normal cervix, suggesting that CIN is associated with a dysfunctional proliferation of cervical epithelium, (2) that there is a significant correlation between the PCNA grade and CIN grades, and (3) the 'mitosis grades' have a strong correlation with the CIN grades.

The purpose of this study is 1) to examine the presence of the antigen in serum by employing a newly developed ELISA immunoassay that quantifies the total antigen and bound antigen (antigen-antibody complex) using polyclonal chicken antibodies directed against an IgM-binding epitope of the new antigen, and 2) to determine the location of the antigen in carcinoma and normal cells. Sera from healthy volunteers (n = 147) and cancer patients (n = 26) were compared for both bound and total antigen concentrations using the new ELISA. Healthy volunteers were subdivided into three groups: those with a personal history of cancer (n = 13), those with no personal or family history of cancer (n = 36) and those with a family history of cancer (n = 97). Ovarian, breast, colon carcinoma tissues and their normal counterparts and cultured ovarian and prostatic carcinoma cells were subjected to immunofluorescence using IgY antibodies and goat anti-chicken fluorescent secondary antibodies. Basic imaging was performed on tissue sections while confocal microscopy was performed on cultured cells. Furthermore, immunohisto-chemical staining using an anti-chicken HRP-conjugated secondary antibody was performed on 16 normal ovarian tissues, 53 ovarian adenocarcinomas, and 3 borderline ovarian tumors. Statistical analysis revealed significant differences in cancer patients' bound and total antigen levels compared to that of healthy volunteers (p < 0.005). Bound and total antigen levels of cancer patients were also significantly higher than those of the healthy volunteers with no personal or family history of cancer and those with a family history of cancer (p < 0.01). However, no significant difference existed between the bound (p > 0.120) and total antigen levels (p > 0.076) of cancer patients and patients with a personal cancer history. Immunohistochemical staining of ovarian tissues revealed a significant difference in the lumenal staining of the carcinomas compared to that of the normal ovarian tissues. Furthermore, fluorescence imaging revealed that the antigen is localized to the cell membranes of the carcinoma cells but is absent from the normal tissues. Confocal microscopy further emphasized the antigen's association with the membrane and also revealed some filamentous cortical staining.

Natural killer T cells (NKT cells) are a subset of T cells. They regulate a wide range of diseases including infection, tumor growth, and autoimmune diseases, through recognizing glycolipid antigens in the context of CD1d. An understanding of the scope of glycolipid antigens would facilitate use of this cell type in controlling immune responses. Till today, a lysosomal glycolipid, isoglobotrihexosylceramide (iGb3), is the only natural glycolipid that has been found to be recognized by both human and mouse NKT cells. To elucidate the molecular basis of this specific recognition, iGb3 variants were designed and prepared: i) replacement of the C26 acyl chain with shortened acyl chains; ii) replacement of the distal galactose with glucose and mannose; iii) replacement of the intermediate galactose with glucose; iv) replacement of the proximal glucose with galactose. Among these glycolipids, the iGb3 variants with shortened acyl chains are potent stimulators of NKT cells. The iGb3 variant with intermediate glucose also showed the ability to stimulate NKT cells, but this finding needs to be verified. Our findings support the specific recognition of iGb3 by NKT cells. The search for other natural glycolipid antigens focuses on glycolipids that are isolated from bacteria and parasites. Recently, glycosphingolipids (GSL-1, -3, and -4) isolated from the sphingomonodaceae family of bacteria were characterized. GSL-1 has been shown to be a potent stimulator of NKT cells. Moreover, it has been reported that GSL-4 is a stimulator as well. To verify the structures and stimulatory properties of GSLs, GSL-1 to -4 were prepared and tested for their abilities to stimulate NKT cells. The result that only GSL-1 can stimulate NKT cells suggests that synthesis of these higher order GSLs would be an immune evasion mechanism. Neutral glycosphingolipids from sheep-derived F. hepatica liver flukes, a causative agent of fascioliasis, were isolated and characterized. Their structures are closely related to iGb3. Among these glycolipids, neo-iGb4s could be truncated to iGb3 in the lysosome and thus stimulate NKT cells. To test this hypothesis, these glycosphingolipids were prepared and tested. None of these synthetic glycolipids stimulates NKT cells, which suggests that the secretion of these glycolipids by F. hepatica could be the result of the parasite-immune-evasion mechanism.

Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.

In Ratten und Mäusen aktiviert der superagonistische anti-CD28 monoklonale Antikörper (CD28SA) vorzugsweise regulatorische T-Zellen. In niedriger Dosierung führt CD28SA zu einer fast ausschließlichen Aktivierung von regulatorischen T-Zellen (Tregs). Diese Beobachtung konnte inzwischen auch für menschliche Zellen in Zellkultur bestätigt werden. In gesunden und freiwilligen Testpersonen deutet die Zytokin-Antwort nach Applikationen von niedrigen CD28SA-Dosen darauf hin, dass sich diese Beobachtung auch in-vivo bewahrheitet. Eine Gabe von CD28SA in niedriger Dosierung, die zu einer exklusiven Aktivierung von regulatorischen T-Zellen führt, könnte somit in der Behandlung von Autoimmunkrankheiten oder von entzündlichen Erkrankungen eingesetzt werden. Eine mechanistische Erklärung für dieses Phänomen blieb lange Zeit unklar. Die CD28SA-vermittelte T-Zell-Aktivierung ist abhängig von der Verstärkung von basalen tonischen Signalen, die T-Zellen über ihren T-Zell-Rezeptor erhalten. Diese Tatsache führte zu der Hypothese, dass die schwachen, tonischen Signale, die konventionelle CD4+ T-Zellen in Abwesenheit ihrer spezifischen Antigene über den T-Zell-Rezeptor erhalten, ein stärkeres CD28 Signal für ihre Aktivierung benötigen als die selbstreaktiven regulatorischen T-Zellen, die ein stärkeres Selbstpeptid-TCR Signal erhalten. In dieser Arbeit konnte gezeigt werden, dass die Blockade von MHC-Klasse-II-Molekülen in Mäusen, in-vitro und in-vivo, den Vorteil der regulatorischen T-Zellen gegenüber den konventionellen T-Zellen bezüglich der Antwort auf niedrige CD28SA Dosierungen, aufhebt. / In rats and mice, CD28 superagonistic mAb (CD28SA) preferentially activate regulatory T-cells, resulting in near exclusive Treg activation at low CD28SA doses. This observation has recently also been extended to cell culture studies in humans, and the cytokine response of healthy volunteers to low-dose CD28SA application suggests that it also holds true in vivo, and thus can be utilized for the treatment of autoimmune and inflammatory diseases. A mechanistic explanation for this phenomenon, however, remained uncertain for a long time. Given that CD28SA-mediated T-cell activation depends on the amplification of basal tonic TCR signals, the hypothesis was tested that the weak tonic TCR signals received by conventional CD4 T-cells in absence of their cognate antigen require more CD28 signalling input than the stronger TCR signals perceived by self-reactive regulatory T-cells. The experiments of this thesis provide strong evidence that in mice, blockade of MHC class II in vitro or in vivo abrogates the advantage of Treg over Tconv in the response to low CD28SA doses.