Bibliography: leaves 169-208 / ix, 208 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1988

Bacteroides melaninogenicus strains 2D and K110 were characterized with regard to their pathogenic, collagenolytic, proteolytic, hemagglutinating and metabolic activities. Both strains were members of the subspecies 13. melaninogenicus ss. asaccharolyticus. They possessed a cell-bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygen-sensitive hemagglutinin (HA), and a protease. Both strains produced butyric and phenylacetic acids and were infective in guinea pigs as characterized by their ability to produce necrotic lesions and to be transferred from one animal to another. Strain 2D required hemin for growth and its growth rate was influenced by the addition of free amino acids to the medium. The hemagglutinating and proteolytic activities of strain 2D were investigated further to determine their relationship to infection. The soluble HA was reversibly inhibited by Hg and activity was restored in the presence of reducing agents. Iodoacetic acid caused irreversible inhibition. The HA was sensitive to heat and pronase treatment. Treatment of the red blood cells (RBC) with neuraminidase enhanced HA activity while the presence of galactose in the reaction mixture inhibited it, suggesting the involvement of galactose residues on the RBCs in the reaction.. Adsorption of the HA to RBC followed by elution and gel filtration resulted in the recovery of 50% of the HA activity and a 52-fold purification. Protease production by _B. melaninogenicus strain 2D was dependent on the growth rate of the organism. The protease was reversibly inhibited by HgCl₂ and irreversibly inhibited by iodoacetamide and iodoacetic acid. The enzyme was insensitive to serine protease inhibitors and EDTA. The pH optimum for proteolytic activity was 7.0, which correlates with the pH of its natural environment, the gingival crevice. It is thus classified as a neutral sulfhydryl enzyme. A 774-fold purification of the cellular protease of 2D, with a 160% recovery of activity, was accomplished by precipitation with 60% ethanol, ultracentrifugation and gel filtration through Sephadex G-100 and Sepharose 2B in the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four distinct bands, each of which was shown to be associated with carbohydrate. In the absence of SDS only one band, which did not migrate into the gel, was obtained. Any attempts to further dissociate the protease resulted in the loss of activity. The protease was active against azocoll, azocasein, casein and N,N-dimethylcasein. No glycosidase, lipase, collagenase or HA activities were detected. Protein, carbohydrate and lipid were detected in the preparation. The soluble protease which amounted to 20% of the cellular protease of strain 2D was subjected to gel filtration on Sephadex G-100 and eluted in a single peak at the void volume. The properties of the soluble protease were identical to those of the cell associated enzyme, suggesting the presence of a single proteolytic enzyme which was released into the culture medium with cell lysis or due to shedding of outer membrane fragments. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA

Lectins (hemagglutinins) are a type of proteins that could recognize different sugar structures and specifically initiate reversible binding with them. Though they have been universally found in a variety of organisms, they are exceptionally abundant in legumes. From the initial finding of agglutinating red blood cells to the discovery of recognizing carbohydrates on cell membranes, multiple functions of lectins have been gradually unveiled by numerous researchers across a century. Based on its carbohydrate-binding property, lectins have found great value in the study of glycomics. Many lectin-based biological tools, like lectin affinity chromatography, lectin blotting, lectin histochemistry, lectin microarray and lectin-based biosensor have been developed and applied to the study of glycoproteins. Besides, lectins are also reported to be potential agents for anti-insect, anti-fungi, anti-HIV, anti-bacterial and anti-tumor applications. / The present study focuses on the isolation of a new hemagglutinin from an edible legume, exploration of its anti-colorectal cancer effect and mechanisms, its cytokine inducing function and anti-HIV activities. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-Gel Blue Gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion-dependent and stable over a wide range of temperatures (20-60℃) and pH (2-11) values. Like most of the lectins or hemagglutinins, this novel hemagglutinin could also attenuate the activity of HIV-1 reverse transcriptase. / This hemagglutinin could potently suppress the proliferation of colorectal carcinoma HCT116 cells and colorectal adenocarcinoma HT29 cells. It induced cell cycle arrest in G0/G1 phase, downregulated the expression of Cyclin D1 and upregulated P21expression. The protein initially bound on the cell membranes most probably through glycoproteins and subsequently entered the cytoplasm, which was achieved as early as 3h post treatment. The hemagglutinin was found to be preferentially localized in Golgi apparatus and initiated aggregation of the Golgi apparatus, which may possibly attenuate its protein processing capacity by reducing total superficial area or even partially blocking the transportation of proteins from the endoplasmic reticulum (ER). The impaired protein reception ability of Golgi apparatus may lead to the protein accumulation in the ER and induce cell apoptosis. Accordingly, two ER stress sensors (IRE1α and ATF6) and one late product of ER stress (CHOP) were found to up-regulated. Apoptosis-inducing effect of this hemagglutinin on HT29 and HCT116 cells were further confirmed using methods based on different principles. Cells treated with the hemagglutinin were observed to undergo obvious chromatin condensation, mitochondrial membrane depolarization and phosphatidylserine exposure. An apoptosis initiator (Apaf-1) and one important indicator (cleaved PARP) of cell apoptosis were accordingly detected. Besides, intraperitoneal administration of this hemagglutinin to colorectal tumor bearing nude mice could slow down the growth of tumors. / At last, this hemagglutinin exerted an immunomodulatory function on splenocytes by stimulating the mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), interferon- gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α). Secretion of IL-1β and IL-2 from splenocytes also increased with the concentration of this hemagglutinin. / In a short conclusion, we have isolated a new hemagglutinin with anti-HIV RT, anti-colorectal cancer and immunomodulatory activities. / 凝集素（血凝素）是一类能够识别不同糖结构并能和它们发生可逆性结合的蛋白。虽然他们在许多生物体内均有发现，但这类蛋白在豆科植物中的含量尤其丰富。经过一个多世纪来众多研究者的努力，从最初认识到其具有红血细胞凝集功能到糖类识别作用，凝集素的诸多功能已被逐步挖掘。基于其独特的糖结构识别特性，凝集素在糖组学的研究中具有重大意义。许多基于凝集素的生物方法，如凝集素亲和层析法，凝集素印迹法，凝集素组织化学，凝集素生物芯片以及基于凝集素的生物传感器已被研究出来, 并用于研究糖蛋白。除此之外，研究表明，凝集素还具有抗虫，抗真菌，抗HIV，抗细菌和抗癌等活性。 / 该凝集素可以极大抑制结肠直肠癌HCT116细胞和结直肠腺癌HT29细胞增殖，引发细胞周期停滞，分别下调和上调Cyclin D1和P21的表达。该蛋白极有可能首先通过和细胞表面的糖蛋白结合而附在细胞膜上，然后进入细胞内。该过程可在往细胞培养液内加入该蛋白后的3小时内完成。该凝集素优先与细胞内的高尔基体结合，随后引发高尔基体聚集。该聚集作用可能会通过减少高尔基体总表面积甚至阻塞内质网和高尔基体间的蛋白运输，进而减弱高尔基体处理蛋白质的能力。当高尔基体接受蛋白的能力降低时，蛋白可能会堆积在内质网上并进一步引发细胞程序性死亡。相应地，两个内质网应激感受蛋白IRE1α和 ATF6以及内质网应激后期产物CHOP均被发现上调。该凝集素对HT29细胞和HCT116细胞的凋亡诱导作用采用不同的方法进行了进一步的确认，这些方法都是基于不同检测原理进行的。结果表明，该凝集素可导致细胞产生明显的染色质凝缩，线粒体膜电位去极化和磷脂酰丝氨酸外翻。与此相应地，凋亡启动蛋白Apaf-1和凋亡后期蛋白(被剪切的PARP)可在处理后的细胞中检测到。通过腹腔注射的方法给接种大肠癌细胞的裸鼠给药可降低肿瘤的生长速度。 / 本研究的工作包括：从一种可食用豆类中提取一种新的凝集素；检测其抗大肠癌的作用和机制；研究其细胞素诱导作用以及抗HIV活性。该蛋白采用液相色谱法分离提纯，其中包括亲和层析柱Affi-Gel Blue Gel, 离子交换层析柱Mono Q 和凝胶层析柱Superdex 75，后两种层析法在FPLC系统上操作。该蛋白的红血细胞凝集作用具有金属阳离子依赖性，并在20-60℃和pH2-11范围内保持活性稳定。像许多其它的凝集素一样，该蛋白也可以削弱HIV逆转录酶活性。 / 最后，该蛋白还具有免疫调节作用，它可促进白细胞介素-2，白细胞介素-6，白细胞介素-1β，干扰素-γ和肿瘤坏死因数-α在mRNA水平上的表达并刺激白细胞介素-2和细胞介素-1β的分泌。 / 综上所诉，本研究分离提纯了一种新凝集素，它具有抗HIV，抗大肠癌和免疫调节作用。 / Dan, Xiuli. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 153-170). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Influenza virus is a pleiomorphic human pathogen which causes self-limiting respiratory illness lasting one-two weeks in most individuals. However, in immunologically compromised individuals, influenza infection may lead to severe morbidity and fatality. Annual epidemics cause 250,000-500,000 deaths worldwide and remain a major public health threat. The virus has evolved mechanisms of antigenic ‘drift’ and ‘shift’ to evade the host immune response. Hence, current influenza vaccines need to be updated every few years. Moreover, the currently available inactivated/live attenuated vaccines entail virus culture in embryonated chicken eggs hindering rapid scale-up. The aforementioned limitations of the current vaccines has had debilitating effect when strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past, despite intensive monitoring. Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift, facilitating a potential pandemic outbreak. These concerns have expedited efforts towards developing a ‘universal’ flu vaccine. Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the mature, disulfide linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit while the stem domain predominantly comprises of the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH induced conformational rearrangement in the host-cell endosomes to adopt the virus-host membrane fusion competent state. The ‘antigenic sites’ on the immunodominant globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed neutralizing antibodies (nAbs). As opposed to the highly-variable head domain, the HA stem is conserved and targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes. Although several bnAbs bind to the conserved HA stem, focusing the immune response to this conserved, subdominant stem domain in presence of the variable head domain of HA has been challenging. Alternatively, mimicking the epitope of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogen capable of eliciting a broadly protective immune response has been difficult because of the metastable conformation of HA. Addressing the aforementioned challenges, we describe the design and characterization of novel influenza immunogens by HA protein minimization. Chapter 1 gives an overview of the influenza virus life cycle, and outlines the structural organization and function of viral proteins. The conventional vaccines that are currently used and their limitations are described in this chapter. Recent improvements in influenza vaccine production focusing on recombinant HA as an alternate solution are discussed. Painstaking efforts of several groups in the recent past has led to the isolation of bnAbs that recognize novel ‘antigenic signatures’ within the globular head and the HA stem domains. Attempts to focus the immune response to these ‘cross-protective’ epitopes are described. The design and characterization of trimeric HA stem-fragment immunogens from influenza A Group-1 viruses which mimic the native, pre-fusion conformation of HA are described in Chapter 2. We engineered ‘headless’ HA stem immunogens based on influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation sensitive stem-directed bnAbs such as CR6261, F10 and FI6v3 with high affinity (equilibrium dissociation constant [KD] of 10-50nM). The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both Group-1 (H1, H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD90) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens conferred robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous HA stem domain immunogens, the designed immunogens described here were purified from the soluble fraction in E.coli. These HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks. The detailed mechanism(s) by which our ‘headless’ stem immunogens provide protection need further investigation. The long central α-helices (LAH) located in the HA stem assemble together into a parallel, trimeric coiled-coil. Immunization with the wt-LAH (76-130 of HA2) derived synthetic peptide designed from an H3 subtype (H3N2 A/Hong Kong/1/68) and conjugated to keyhole limpet hemocyanin (KLH) was shown previously to elicit antibodies reactive in ELISA with multiple hemagglutinin subtypes and to confer protection against challenge with H3N2, H1N1 and H5N1 virus strains. The LAH peptide sequence was chosen based on maximal binding to the monoclonal antibody (MAb), 12D1, which has broad neutralizing activity against influenza viruses of the H3 subtype. These results motivated us to rationally design stabilized derivatives of wt-LAH and test their protective capacity in a mouse challenge model of influenza. This work is described in Chapter 3. Additionally, to understand the contribution towards protection conferred by the two distinct surface exposed patches on LAH, we designed constructs spanning different stretches of LAH. The biophysical characterization of the LAH-derived constructs indicates that most of them were well-folded. All these constructs were moderately immunogenic in mice but at best, conferred limited protection from lethal viral challenge. In contrast to previously reported results, our data suggests that the LAH in the absence of other regions of HA may require not only strong, but also specific adjuvantation to induce a robust and functional immune response in vivo. Chapter 4 describes an immunogen design (H1pHA9) based on the globular head domain of pandemic H1N1 HA which can be produced using a prokaryotic expression system. The HA-fragment, H1pHA9, stably refolds to mimic the conformation sensitive neutralizing epitopes in the globular head domain of HA. We have also successfully engineered the HA head domain to delineate the epitope of antibodies neutralizing the pandemic H1N1 virus using a yeast cell-surface display platform. In this direction, we report the isolation of a novel, neutralizing murine MAb, MA2077, against the pandemic H1N1 virus. The epitope of this MAb has been mapped onto the ‘Sa’ antigenic site. The ability of the head domain fragment, H1pHA9, which binds MA2077 with high affinity to elicit such neutralizing antibodies in vivo needs to be further explored. Structural analysis has shown that elements of the HA stem diverge between the two phylogenetic groups. Therefore, to mitigate the threat of circulating influenza A viruses from these distinct structural classes (H1 and H3 belonging to Groups 1 and 2 respectively), in lieu of a ‘universal’ vaccine, a combination of immunogens derived from both the groups is a practical alternative. In Chapter 5 we describe the design of stem-fragment immunogens from an influenza A Group-2 virus strain. We report the characterization of engineered ‘headless’ HA stem immunogens based on influenza A/Hong Kong/1/68 (H3N2) subtype. The designed immunogens were expressed in E.coli and purified from the soluble fraction with abundant yields (~15mg/lt). The HA stem-fragment immunogens could be concentrated to high concentrations without aggregation. While, H3HA10-IZ and H3HA10-Foldon, the trimeric derivatives of our parent construct (H3HA10) which were folded, conferred modest protection against a lethal homologous virus challenge in mice, there is considerable scope to improve our immunogen design. Analyzing the results from our previous work (Chapter 2), we speculate that structural elements at the N-terminus of A-helix are critical for helix initiation. We therefore extended the design to include residues from the start of the A-helix. We designed the extended stem immunogens from both H3 and H7 subtypes. The proteins were purified from the soluble fraction of the E.coli cell culture lysate. Preliminary studies suggest that extension of the A-helix has aided proper folding. These proteins need to be further characterized and evaluated in an animal model.

Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

Bakgrund: Individer i ABO-systemets grupp O saknar antigen A och B på de rödablodkropparna, vilket medför att blodet kan transfunderas över blodgruppsgränserna.Förekomsten av anti-A och anti-B-antikroppar, så kallade hemagglutininer, i grupp Oplasmanförsvårar dock transfusion av blodkomponenter innehållandes plasma, eftersomdessa kan orsaka en hemolytisk reaktion där mottagarens röda blodkroppar förstörs. För attminska risken för komplikationer bestäms koncentrationen av antikroppar. Det är känt attkoncentrationen av hemagglutininer kan förändras över tid beroende på olika faktorer, vilketdock inte tas hänsyn till i praktiken. Det är ett outforskat område kring huruvida en individshemagglutininer kan reagera olika med olika individers erytrocyter. Syfte: Syftet var att undersöka om koncentrationen av hemagglutininer kan variera över tid,samt variera i reaktionsstyrka med olika individers röda blodkroppar. Metod: Metoden var en laborativ metod enligt rådande praxis i Region Örebro Län,användande av gelkortsteknik efter spädning av plasmaprov. Gelkortstekniken går ut på attplasma, spädd till förbestämda gränsvärden (IgM <1:100 och antikropp typ IgG <1:400)blandas med röda blodkroppar i mikrorör vilket ger en visuellt avläsbar reaktion om det finnsantikroppar i plasmaprovet. Resultat: Vid användning av rådande metod för antikroppsbestämning ses stora variationerantikroppskoncentration både över tid och mot olika röda blodkroppar. Av 8 tidigare högtitrigaplasmaprover var 5 stycken fastställt hög-titriga efter upprepade undersökningar. Ävenför de prover som gav kraftigast reaktioner sågs stora skillnader mot olika erytrocyter. Slutsats: Relaterat till den stora spridningen i antikroppstiter av hemagglutininer som ses vidundersökning mot olika testerytrocyter, är rådande praxis för kvantifieringsbestämning avanti-A och anti-B inte optimal. Det behövs forskning som tar fram en standardiseradtesterytrocyt och fastställer evidensbaserade gränsvärden för kritiska titrar.

Xia Lixin. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 210-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

La transmission des virus grippaux de type A s’effectue via l’eau, l’air ou les surfaces. Elle implique donc toujours une étape dans l’environnement, durant laquelle les virus sont inactivés plus ou moins rapidement en fonction du sous-type ou de la souche virale analysés. Cependant, à ce jour, les facteurs moléculaires déterminant la stabilité des particules virales en dehors de l’hôte restent largement méconnus. Dans le but d’identifier ces déterminants, nous avons généré différentes combinaisons de réassortiments entre deux virus grippaux de sous-types H1N1 possédant un phénotype de stabilité différent. Les stabilités respectives de ces virus réassortants ont été évaluées dans un environnement-modèle, puis comparées entre elles. Pour cela, nous avons utilisé un système d’analyse en temps réel des cultures cellulaires, permettant de calculer, pour chacun des virus testés, une pente d’inactivation moyenne et, in fine, de mesurer l’influence respective de chacun des segments viraux sur le phénotype de stabilité des virus. D’après nos résultats, le phénotype de stabilité des virus grippaux est majoritairement déterminé par l’hémagglutinine (HA) et la neuraminidase (NA), qui sont les principales glycoprotéines de surface de ces virus. De plus, nous avons identifié des changements d’acides aminés dans la HA et dans la NA, qui ont pour effet une diminution ou une augmentation de la stabilité des particules virales dans l’environnement. Nous avons également montré qu’un virus avec un gène de la HA codons-optimisés, et donc porteur de mutations synonymes, suffit pour augmenter significativement la stabilité des particules virales dans l’environnement. La stabilité de la HA à pH acide, le taux d’expression de la HA dans les cellules infectées, et le nombre de sites de fixation aux ions calcium dans la NA sont modifiés par les mutations décrites dans cette étude, et sont donc des facteurs de stabilité des particules virales. De plus, une analyse en microscopie a permis de montrer que les virus inactivés dans l’environnement peuvent fixer leurs récepteurs cellulaires, mais sont incompétents pour induire l’étape de fusion dans l’endosome nécessaire à l’entrée des virus dans la cellule. Ces deux étapes du cycle viral sont dépendantes de la HA. Dans l’ensemble, nos résultats montrent l’importance de la HA et de la NA des virus grippaux dans la détermination du phénotype de stabilité des virus grippaux dans l’environnement. Par conséquent, la diversité connue des HA et NA dans la nature laisse supposer des variations fréquentes du phénotype de stabilité de ces virus. Leur étude pourrait permettre de mieux décrire l’écologie et l’épidémiologie de ces virus. L’analyse des données épidémiologiques et climatiques des épidémies de grippe saisonnière, sur 5 ans et dans 13 pays, a ainsi révélé une différence de distribution des virus H1N1 et H3N2, en fonction de la température hebdomadaire dans ces pays. La comparaison de la stabilité de ces virus sur des surfaces, à 4 °C et à 20 °C, suggère que la distribution des sous-types viraux au début des épidémies est en partie régulée par leur stabilité en fonction de la température / The transmission of Influenza A viruses (IAV), either airborne in mammals or oro-faecal in aquatic birds, submits viral particle to a wide range of environmental conditions. These environmental conditions modulate IAV survival outside the host, which is also dependent on the viral subtype or strains. To date, the molecular drivers of IAV environmental persistence remain to be identified. In order to identify IAV molecular drivers of the environmental persistence, we generated different reassortant viruses between two H1N1 viruses that do not have the same stability outside the host. To this purpose, we performed survival kinetic and compared the inactivation slope of generated reassortant viruses in our controlledenvironment, using a real time cell analysis system. Our results demonstrate that the hemagglutinin (HA) and the neuraminidase (NA) are the main viral segments driving IAV environmental persistence. In addition, mutations driving viral stability in the environment were identified in the HA and NA amino-acid sequences. We also demonstrated that synonymous mutations introduced in the HA, using a codon-optimization strategy, drive the environmental persistence of IAV. The HA stability at low pH, HA surface expression levels in infected cells and the number of calcium binding sites of the NA were alternately changed by the mutations described in our study, indicating that these are stability determinants of IAV survival outside the host. Then, the sequential events of viral entry were analysed with fluorescence microscopy assays, showing that viral particles being exposed for a long period in saline water at 35°C are still able to bind their cellular receptor whereas the HA-mediated fusion within the endosome is not possible anymore. These two steps of the viral cycle are mainly mediated by the HA protein. Altogether, these result highlight the importance of the HA and the NA proteins, driving the environmental persistence of IAV. Given the known diversity of these two proteins in nature, this arouses interest in studying IAV environmental persistence at a more global scale. Such study could improve our knowledge on IAV ecology and epidemiology. Epidemiologic and climatic data analyse of human seasonal influenza viruses during 5 years and from 13 countries revealed that H1N1 virus and H3N2 virus distribution differs according to the mean weekly temperature in these countries. We then compared the H1N1 virus and H3N2 virus persistence on stainless steel surface at 4 °C and 20 °C, and the preliminary results suggest that IAV seasonal subtypes distribution might be partly regulated by their stability according to the temperature

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.