Dissertations / Theses on the topic 'Vaccine candidate protein'

Francisella tularensis is one of a small group of bacteria recognized for their virulence and potential for use as biological weapons. In this study we utilize a novel approach to identify an immunologically prominent component of F. tularensis that appears to be a promising vaccine candidate. Francisella is an intracellular pathogen that infects cells of the reticuloendothelial system. Other bacteria, such as Brucella spp. have this part of their life cylce in common. However, while mice injected with Brucella spp. survive and produce antibodies to the bacteria which are immunologically reactive not only with Brucella spp. but, also with Francisella. When we vaccinated mice with a B. abortis O-linked polysaccharide (OPS) and then challenged them with 10 LD50F.tularensis LVS, 60% survived. Sera from Brucella OPS-primed/F.tularensis-challenged mice was used to identify immune reactive proteins from F. tularensis. A novel 52 kDa fraction was identified. While vaccination of mice with this partially purified fraction only provided 20% protection to F.tularensis challenged mice, both whole cell extracts and a partially purified soluble fraction (>30kDa) given to Brucella-vaccinated mice were 100% protective. The 52 kDa enriched fraction elicited a rudimentary cytokine burst of nitric oxide in a cell culture of J774.1 macrophages. The 52 kDa fraction was degraded by proteinase K and appeared to decrease in size to 36 kDa in the presence of DNAase, suggesting a possible protein and nucleic acid composition. The host response to F. tularensiss infection is complex, but given the ability of the 52 kDa component to protect against live vaccine challenge, and its apparent ability to elicit a cytokine burst, this component may have potential use in future vaccine production.
xii, 97 leaves ; 29 cm.

The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.

Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Los virus de influenza A (IAVs) son los responsables de brotes pandémicos y de la mayoría de las epidemias anuales en los seres humanos, aves y cerdos. Los virus de influenza A se dividen en subtipos, basado en la naturaleza de sus glicoproteínas de superficie, hemaglutinina (HA) y neuraminidasa (NA). La HA es una glicoproteína de superficie homotrimérica que media la entrada del virus de influenza a través de la unión celular y eventos de fusión de membranas. El bolsillo de unión al receptor de HA está rodeado por los sitios de unión de anticuerpos antigénicamente variables. Por lo tanto, los anticuerpos limitados a estos sitios debería, en principio, bloquear la unión a las proteínas del receptor, inhibiendo la entrada del virus, lo que demuestra actividad de inhibición de la hemaglutinina y la actividad de neutralización viral. Sin embargo, la subunidad 1 de HA (HA1) es altamente variable a través de los virus de influenza y tiende a cambiar bajo presión inmune; por lo tanto, fácilmente evade los anticuerpos neutralizantes inducidos por la vacunación o infecciones anteriores. Las consecuencias negativas de las infecciones por virus de influenza incentivan al mundo de la ciencia para promover el desarrollo de vacunas multivalentes contra la gripe, que tengan eficaz protección frente a todas las cepas de influenza. Por consiguiente, el campo de la bioinformática se ha convertido en una parte importante de la identificación y validación temprana de nuevas dianas terapéuticas que podrían ser un primer paso esencial en el desarrollo de una vacuna eficaz para el virus de influenza, que representa la alta variabilidad de sus determinantes antigénicos. En esta tesis se postuló que la HA1 podría representar una diana potencial para una vacuna multivalente frente a la infección por influenza virus. Por ende; el objetivo general de esta tesis fue seleccionar péptidos conservados de la subunidad 1 de la proteína hemaglutinina del virus de la gripe, para evaluar la eficacia de los candidatos seleccionados, para inducir la inmunidad que pueda proteger a los animales contra la infección. Para desarrollar este objetivo, se llevaron a cabo tres estudios experimentales: en ratones (Capítulo 1), cerdos (capítulo 2) y pollos (Capítulo 3). En el primer estudio, se evaluó el efecto protector de la mejora de péptidos HA1 contra la pandemia del virus H1N1 2009 (pH1N1) y el virus de la influenza de alta patogenicidad H7N1 en un modelo de ratón (Capítulo 1). En este estudio, los ratones fueron vacunados por vía intraperitoneal con la mezcla de péptido sintético (NG34 + DC89) y desafiados ya sea con el pH1N1 ó H7N1 del virus de influenza. Una mortalidad del 85% fue observada en los ratones de control, independientemente del virus utilizado para el desafío, 80% y 66% de los ratones vacunados con péptido sobrevivieron al desafío con pH1N1 y H7N1, respectivamente, sin la detección de virus de la influenza (IV). Los ratones vacunados supervivientes se correlacionaron con la presencia de anticuerpos neutralizantes de reactividad cruzada en sueros antes de la exposición. La inmunización con NG34+DC89 también indujo respuesta inmune en mucosas; demostrándose con la presencia de IgA en lavado broncoalveolar en el 50% de los animales. Nuestros resultados también muestran que la vacunación de NG34+DC89 es capaz de inducir anticuerpos y la protección cruzada neutralizante frente a dos cepas heterólogas, pH1N1 y H7N1. Por lo tanto, péptidos NG34+DC89 representan un inmunógeno atractivo, lo que podría ser aún más optimizado para futuras formulaciones de vacunas multivalentes frente el virus de influenza. En el segundo estudio, hemos probado la inmunogenicidad de un cóctel HA1-péptido en un modelo porcino para evaluar si esta nueva formulación puede conferir inmunidad a una amplia gama de virus de influenza in vitro (Capítulo 2). En este estudio, hemos utilizado cuatro péptidos de la HA1 del subtipo H1 del virus de influenza (NG34, DC55, RA22 y SS35), evaluamos su inmunogenicidad en cerdos convencionales de granja frente a virus de influenza homólogos y heterólogos. Las vacunas de péptidos sintéticos indujeron anticuerpos con capacidad neutralizante y de inhibición frente a virus homólogos. Aquellos también cros-reaccionaron frente a los virus heterólogos de origen aviar como el virus H7N1 y virus de influenza de baja patogenicidad H5N2 y además el virus circulante H3N2 de origen porcino. Por otra parte, se detectaron anticuerpos específicos de IgA-secretora en hisopos nasales. Los resultados muestran que los péptidos evaluados fueron inmunogénicos en cerdos. La respuesta humoral con la actividad neutralizante y de inhibición de hemaglutinina generada después de la inmunización podría ser utilizada en otros estudios de inmunidad protectora heterosubtípica. En el tercer estudio se evaluó el efecto protector de la mejora de péptidos de HA1 contra el virus altamente patógeno virus de la influenza H7N1 en pollos, un modelo huésped natural (capítulo 3). En este estudio, se utilizaron dos péptidos sintéticos NG34 y SS35 para vacunar pollos de granja. La vacunación tanto con péptidos NG34 ó SS35 indujeron anticuerpos específicos que reconocen virus heterólogos, como H7N1 y H5N2 in vitro. La vacunación con péptido NG34 provocó una respuesta de anticuerpos protectores que confirió una protección parcial frente un desafío letal con H7N1. Además, el péptido NG34 indujo una respuesta inmune de la mucosa, que correlacionaba con la diseminación viral reducida en hisopos orofaríngeos/ cloacales y la pulpa de la pluma. Por el contrario, animales vacunados con el péptido SS35 no pudieron producir una eficiente respuesta inmune protectora frente el desafío letal H7N1. Es necesario recalcar, que todos los péptidos HA1 del subtipo H1 del virus de influenza, fueron seleccionados mediante el Método de espectros informativo (ISM). Cuatro principales conclusiones generales pueden extraerse de estos estudios: (i) Péptidos HA1 son inmunogénicos en todos los modelos animales estudiados (ratones, cerdos y pollos) e inducen una respuesta immune humoral y de mucosa. (ii) Péptidos inmunogénicos de la subunidad 1 de la proteína hemaglutinina del virus de la influenza confieren una protección parcial contra diferentes subtipos virales en ratones; (iii) Los cerdos vacunados con péptidos HA1 provocan una respuesta de anticuerpos neutralizantes con actividad de inhibición de la hemaglutinina frente a diferentes subtipos de Influenza A virus y (iv) Péptidos HA1 confieren una protección parcial contra un virus altamente patógeno/ H7N1 en modelo de pollo. En general, nuestros resultados proporcionan ideas sobre nuevos enfoques para la vacunación de la gripe y la comprensión de la respuesta inmune frente al virus de influenza en ratones, cerdos y pollos.
Influenza A viruses (IAVs) are responsible for pandemic outbreaks of influenza, and for most of the well-known annual flu epidemics, in humans, poultry and pigs. IAVs are divided into subtypes, based on the nature of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The HA is a homotrimeric surface glycoprotein that mediates influenza viral entry via cellular attachment and membrane fusion events. The receptor-binding pocket of HA is surrounded by antigenically variable antibody binding sites. Therefore, antibodies bounded to these sites should, in principle, block the binding to receptor proteins, inhibiting viral entry, demonstrating hemagglutinin inhibition activity and viral neutralization activity. However, the subunit 1 of HA (HA1) is highly variable across viruses and tends to change under immune pressure and, hence, easily evades the neutralizing antibodies induced by previous vaccinations or infections. The negative implications of influenza virus infections push the world to promote the development of multivalent flu vaccines that protect against all human influenza strains. Therefore, the field of bioinformatics has become a major part of the identification and early validation of new therapeutic targets and could be an essential first step in the development of an effective vaccine for influenza virus that represents the high variability of its antigenic determinants. Therefore, in this thesis it was postulated that the HA1 could represent a potential target for a multivalent vaccine of influenza infection. Consequently, the general objective of this thesis was to select conserved peptides from the HA1 of influenza viruses and to evaluate the efficacy of the selected candidates to induce immunity that can protect animal against infection. To achieve this objective, three studies were undertaken in mice (Chapter 1), pigs (chapter 2) and chickens (Chapter 3). In the first study, we evaluated the protective effect of improved HA1-peptides against the pandemic H1N1 2009 virus and a H7N1 highly pathogenic influenza virus (HPAIV) in a mouse model (Chapter 1). In this study, mice were intraperitoneally vaccinated with the peptide mix (NG34+DC89), and next challenged with either the pH1N1 or the H7N1 strain of Influenza virus. Conversely to the 85% mortality observed in control mice, independently of the virus used for challenge, 80% and 66% of the peptide-vaccinated mice survived the challenge with pH1N1 and H7N1, respectively, without detection of influenza viruses (IV). Vaccinated mice surviving correlated with the presence of cross-reactive neutralizing antibodies in sera prior to challenge. The immunization with NG34+DC89 also induced mucosal immune responses demonstrated with the presence of IgA in bronchoalveolar lavage in 50% of the animals. Our results also show that NG34+DC89 is capable to induce cross-neutralizing antibodies and protection against two heterologous IV, pH1N1 and H7N1. Thus, NG34+DC89 represent an attractive immunogen, which could be further optimized for future multivalent vaccine formulations against influenza virus. In the second study, we tested the immunogenicity of a HA1-peptide cocktail in a pig model to assess whether this new formulation can confer immunity to a wide range of IAVs in vitro (Chapter 2). Four peptides (NG34, DC55, RA22 and SS35) within the HA1 from H1 viruses were selected, and evaluated their immunogenicity in conventional farm pigs against homologous and heterologous viruses of influenza. Peptides immunizations induced HA neutralizing and inhibiting antibodies against homologous viruses. Those also cross-reacted against heterologous viruses like H7N1 and H5N2 and, most importantly, the circulating H3N2. Moreover, secretory IgA-specific HA antibodies in nasal swabs were detected. Altogether, the results show that the peptides tested were immunogenic in pigs. The humoral response with hemagglutinin-inhibiting and cross-neutralizing activity generated after immunization could be used in further studies of protective heterosubtypic immunity. In the third study we evaluated the protective effect of improved HA1-peptides against H7N1 highly pathogenic influenza virus (HPAIV) in chickens, a natural host model (Chapter 3). In this study, based on ISM, we selected two highly conserved peptides (NG34 and SS35) of a H1 influenza virus strain and used them to vaccinate free-range chickens. The vaccination with both NG34 and SS35 peptides induced specific antibodies that recognized heterologous viruses, as H7N1 HPAIV and H5N2 Low pathogenic avian virus (LPAIV) in vitro. Vaccination with NG34 peptide elicited a protective antibody response that conferred partial protection against a lethal challenge with H7N1 HPAIV. Furthermore, NG34 peptide induced a mucosal immune response, which correlated with reduced viral shedding in oropharyngeal/cloacal swabs and feather pulp. On the contrary, SS35 peptide vaccinated animals failed to produce an efficient protective immune response as no survival against lethal H7N1 challenge was achieved. Finally, it remains to point out that all HA1-peptides from H1 subtype of influenza virus were selected by the method of informative spectra (ISM). Four main general conclusions can be drawn from these studies: (i) HA1-peptides are immunogenic in all the animals models tested (mice, pigs and chickens) and induce humoral and mucosal immune response. (ii) Novel conserved immunogenic peptides from the hemagglutinin subunit 1 protein of influenza viruses confer partial protection against different viral subtypes in mice; (iii) Pigs vaccinated with HA1 peptides elicit neutralizing and hemagglutination-inhibiting antibody responses against different subtypes of Influenza A virus and (iv) Synthetic peptides from the hemagglutinin of influenza viruses confer partial protection against highly pathogenic A/H7N1 virus in a free-range chicken model. Overall, these data provide insights on new approaches for vaccination in influenza and understanding of the immune response against influenza viruses in mice, pigs and chickens.

The StkF protein of Salmonella enterica serovar Paratyphi A, a putative adhesion-associated protein, was used to develop a candidate adhesin-based vaccine against Salmonella infections, particularly S. Paratyphi A-associated enteric fever. Subcutaneous immunisation of BALB/c mice with recombinant StkF (rStkF) showed that this protein was strongly immunogenic as revealed by the presence of high-titre (1:50,000) anti-StkF antibody in sera from immunized mice. Furthermore, as IgG1 was the major antibody isotype induced, it would appear that rStkF generates a strong Th2-type humoral immune response. The induction of a Th2-dominant immune response was further confirmed by splenocyte-associated cytokine profile analysis which demonstrated greater up-regulation of IL-4 than that of IFN-γ or IL-12-p70. In terms of the protective potential of this antigen, in vitro assays demonstrated that StkF-specific murine antiserum markedly enhanced opsonophagocytosis-mediated uptake of StkF-expressing Salmonella bacteria by human neutrophils. Augmented antibody-specific complement-mediated lysis targeting StkF-expressing Salmonella specifically was also shown. These data suggest a likely direct contribution of StkF-specific antibodies to in vivo killing and clearance of Salmonella and strongly support further investigation of StkF as a potential Salmonella vaccine candidate. BlastN-based interrogation of the NCBI bacterial genome database and PCR investigation of a larger set of strains has shown that the S. Paratyphi A stkF gene and/or the whole stk fimbrial gene cluster is carried by ~1/3 of examined Salmonella serovars. Additionally, bioinformatics and phenotypic characterisation has revealed that the stk fimbrial operon belongs to the chaperone/usher-γ4-fimbrial clade and that it encodes a mannose-sensitive haemagglutinating fimbrial structure. The latter trait is typical of type 1 fimbriae. ELISAs based on rStkF, rStaF and rSipA showed variable sensitivity and specificity for the serodiagnosis of invasive Salmonella infections depending on the particular UK or region-specific cut off applied for each antigen. Further refinement and/or merging of these assays may allow for development of simple and cost effective tests with higher sensitivity and/or specificity than the Widal test. Importantly, despite some minor reactivity with serum from patients with other Gram-negative bacterial infections, the rStkF-based ELISA exhibited no reactivity with serum from patients with dengue fever supporting its potential as a discriminatory diagnostic tool between fevers caused by S. Paratyphi A and dengue virus.

The major merozoite surface protein of Plasmodium falciparum (PtMSP1) is a malaria vaccine candidate antigen. Monoclonal antibodies (mAb) which bind to disulphide-constrained epitopes of PfMSP1₁₉ inhibit parasite growth in vitro and immunisation of animals with recombinant proteins representing this region of the molecule protects animals against challenge infection. In addition, epidemiological data demonstrates that antibody responses to the C-terminus of PfMSP1 are associated with protection against clinical symptoms of malaria. To further evaluate the potential of this antigen as a vaccine candidate, I have measured human immune responses to recombinant proteins representing the C-terminus of PfMSP1. I have found that PfMSP1₁₉ is naturally antigenic in individuals living in a malaria endemic area and that antibody is cross-reactive between the two major allelic sequences of PfMSP1₁₉. However, some individuals consistently remain nonresponsive to PfMSP1₁₉ despite life long exposure. To determine whether this is due to a lack of T cell help, I evaluated T cell responses to PfMSP1₁₉ in malaria-exposed adults. T cell responsiveness to PfMSP1₁₉ is low and appears to be, in part, due to the disulphide-bonded structure to PfMSP1₁₉ which may hinder antigen processing. I have found that antibody responses to PfMSP1₁₉ are associated with resistance to clinical malaria in two populations of children actively acquiring immunity to malaria. However, not all antibody-positive children were protected from malaria, suggesting that the fine-specificity of the antibody response may be important in determining its ability to provide protection against clinical malaria. I was able to demonstrate that malaria immune IgG, affinity purified to PfMSP1₁₉, inhibits parasite growth in vitro and is equally effective against parasites from either PfMSP1 family. This suggests that if protective antibodies could be induced by vaccination, with either allelic form of the protein, infection by all strains of P. falciparum could be controlled.

Thesis (Ph.D.)--Medical University of Ohio, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Garry Cole. Includes abstract. Document formatted into pages: ii, 206 p. Title from title page of PDF document. Title at ETD Web site: Evaluation of two homologous Coccidioides posadasii antigens as recombinantly expressed monovalent, divalent, and chimeric vaccine candidates. Bibliography: pages 75-83, 116-120, 165-169, 185-204.

Presented in this dissertation are proteomic analysis studies focused on identifying proteins to be used as vaccine candidates against Coccidioidomycosis, a potentially fatal human pulmonary disease caused by inhalation of a spore from the soil-dwelling pathogenic fungi Coccidioides posadasii and C. immitis. A method of tandem mass spectrometry data analysis using dual protein sequence search algorithms for increasing the total protein identifications from an analysis is described. This method was utilized in a comprehensive proteomic analysis of cell walls isolated from the dimorphic fungal pathogen C. posadasii. A strategy of tandem mass spectrometry-based protein identification coupled with bioinformatic sequence analysis was used to produce a list of protein vaccine candidates for further testing. A differential proteome analysis using stable isotope protein labeling was undertaken to identify vaccine candidate proteins that are more highly expressed in the spherule, or pathogenic phase, of C. posadasii. The results of these analyses are 9 previously undescribed protein vaccine candidates isolated from spherule cell walls that have sequence indications of extracellular association such as GPI anchors and N-terminal signal sequences and antigen potential based on homology to known antigenic or secreted proteins. An additional 14 proteins identified from spherule cell walls are potential vaccine candidates based on extracellular sequence predictions without any indications of antigenic potential. The stable isotope labeling study has identified 3 more proteins that are preferentially expressed in spherules and exhibit antigenic potential based on extracellular localization or homology to known antigenic proteins. Additionally, there were 5 unknown function proteins identified by stable isotope labeling that are more highly expressed in spherules that may be good vaccine candidates but cannot be identified or localized by sequence analysis.The dual algorithm protein identification method presented here is a new technique to address some common shortcomings associated with a proteomic analysis. The comprehensive proteomic analyses of Coccidioides posadasii presented here have provided new targets for Coccidioidomycosis vaccine development as well as insights into the proteome of this pathogen, such as the sequence comparison of C.posadasii proteins to human proteins, as well as a comprehensive analysis of predicted protein function in the Coccidioides proteome.

Malaria vaccine research has been battling with persistent challenges, including polymorphisms of vaccine antigens, difficulties with production processes, and limited immune protection against the disease. Intrinsically unstructured proteins (IUPs) are a fairly newly classified group of proteins that have no stable 3D structure and are generally heat-resistant. They usually contain low complexity regions and repetitive sequences, both of which are distinct characteristics of the malaria proteome. Surprisingly, some of the vaccine candidates that have been extensively studied were later reported to have unstructured regions, some of which serve as targets of protective immunity. In keeping with their interesting immunological profiles and their unique properties, which are exceptionally beneficial for vaccine production, malarial IUP antigens may be good vaccine candidates. This PhD project has the following aims:- 1) to develop a synthetic unstructured protein antigen based on the Block 2 region of MSP-1, named the MSP-1 hybrid 2) to characterize a novel vaccine antigen derived from the MSP-3.3 protein, namely an IUP region of PF10_0347 gene product, for its potential as a vaccine candidate 3) to develop a second-generation vaccine by combining the MSP-1 hybrid, with two allelic variants of MSP-2, to overcome antigenic polymorphism and strain-specific immune responses 4) to validate protocols for IUP identification from proteins extracted from the malaria parasite. This study showed that 1) MSP-1 hybrid production was scalable, yielding high protein yields with comparable immunological properties to small-scale production. MSP-1 hybrid was shown to be compatible with different adjuvants, and elicited specific antibodies covering the whole range of Block 2 allelic diversities. 2) A novel antigen, MSP-3.3C, an IUP based on the 3’ region of the PF10_0347 gene, was cloned, expressed and purified. Anti-MSP3.3C antibodies showed very strong parasite growth inhibitory effects in vitro. 3) The MSP-multihybrid antigen was expressed using simple techniques, but only at low levels. It contains epitopes from all three parasite antigen components, and is recognized by specific naturally acquired antibodies. 4) an unconventional 2D gel technique was tested as a method of malaria parasite IUP identification. Plans for further validation of this technique were discussed.

Neisseria meningitidis is a major cause of meningitis and septicaemia in infants and children. Disease caused by serogroup A, C, W-135 and Y strains is prevented by vaccination using capsular polysaccharide preparations. For serogroup B strains for which the capsular polysaccharide is poorly immunogenic, antigenic proteins on the bacterial surface are under investigation as promising vaccine candidates. Due to the variability in sequence of antigenic proteins, vaccine formulations containing combinations of proteins and protein variants are tested for broad protection against serogroup B isolates. In search for new members for inclusion in multi-protein vaccines, the surface proteome of N. meningitidis from MC58 strain was investigated using treatment of whole meningococci with proteases followed by quantitative differential in-gel electrophoresis (DIGE) of the meningococcal proteome. The majority of proteins identified included known surface antigens and other uncharacterised predicted surface proteins. Quantitative analysis also allowed the assessment of the extent of protease cleavage on the protein population and suggested proteins were either completely or partially digested. Additionally, differences in the protein content of outer membrane vesicles (OMVs) from H44/76 meningococci grown in rich or minimal culture media were assessed by DIGE. Differentially expressed proteins were related to differences in bactericidal activity detected between immune sera from mice immunised with the different OMV preparations. Of the proteins detected on the surface and OMVs, those able to induce an immune response in mice were identified by immunoproteome analysis. Four of the previously uncharacterised proteins identified by the proteomic analyses were assessed in immunological assays for their potential as vaccine candidates. Recombinant forms of macrophage infectivity potentiator (NmMIP), type IV pili biogenesis proteins PilP and PilO and putative adhesin complex protein NMB2095 were produced in Escherichia coli, purified and used to immunise mice. Immune sera against NmMIP bound strongly to the surface of MC58 whilst exhibiting opsonophagocytic activity and facilitating deposition of complement factors on the surface of various meningococcal strains. The nucleotide sequence encoding NmMIP was assessed across a panel of 106 meningococcal isolates and found to be highly conserved and under negative selective pressure.

Influenza virus causes a highly contagious respiratory disease whose morbidity and mortality is best prevented by vaccination and the need for a rapid and scalable vaccine response to emerging influenza virus strains is widely recognised. In this study, the haemagglutinins (HAs) of human HI and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fe domain. Recombinant HA-human immunoglobulin Fe domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomeric HAs of the anticipated molecular mass, which agglutinated red blood cells and were purified by protein A chromatography. Purified protein was used to immunise mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype- specific haemagglutination inhibition. A degree of heterosubtypic cross reaction between H5 antibodies and HI HA was also shown in this study, plausibly due to structural relatedness of the HAI domains. A Th2 immune response was dominant with some ThI response, typical of the immune responses described for other influenza vaccines and for soluble oligomeric HA. Dose ranging immunisation studies demonstrated significant seroconversion down to 100ng per dose, the human equivalent dose of 24ugs as calculated by body surface area, which is lower than that observed for a recombinant HA in human studies. The polyvalent serum response was directed to the RBS of the HA as reported for experimental influenza infection and included neutralising antibodies. Mutations introduced into the RBS of the H5 HA altered the antigenicity of the virus protein and appeared to elicit antibodies more efficient for binding to human origin influenza subtypes. Epitope specificity studies identified the top of the HAl domain as the target of the induced antibodies, which broadly correlated with the epitope mapping for the H5 HA of several influenza strains. Thus, HuFc-tagged HAs expressed in insect cells are potential candidates for gene-to-vaccine approaches to influenza vaccination.

Malaria Impfstoffkandidaten, welche Sicherheit und Wirksamkeit gegen prä-erythrozytische Stadien bieten, sind nach wie vor in der Entwicklung. Experimentelle Immunisierungsstudien mit genetisch attenuierten Parasiten (GAP), welche die Entwicklung über das klinisch asymptomatische Leberstadium hinaus verhindern, erwiesen sich als sicher und effizient. ΔSLARP GAP-Sporozoiten arretieren vollständig in der Leber, bieten jedoch keinen langanhaltenden Schutz. Hingegen zeigen Immunisierungen mit ΔP36p/P36 Sporozoiten einen langanhaltenden Schutz, führen jedoch während der Immunisierung gelegentlich zu Blutstadieninfektionen. Diese Studie liefert eine systematische vorklinische Bewertung eines dreifachen KO GAP-Parasiten, durch die Kombination von ΔSLARP und ΔP36p/P36. KO Parasiten arretierten vollständig in vitro und in vivo, aber der zeitnahe Blutinfektionsbeginn nach einer Sporozoiteninfektion in Mäusen zeigte eine verminderte Wirksamkeit des Impfstoffs. Während ein besserer Schutz durch einen späten Leberstadien Entwicklungsstillstand erreicht werden kann, bleiben die zugrundeliegenden molekularen Mechanismen unklar. Eine Vorrausetzung für die Leberzellen Antigenpräsentation ist die Präsenz von parasitären Antigenen im hepatozyten Zytoplasma. Der Proteinexportkomplex PTEX ist in Leberstadien nicht vollständig funktionstüchtig, da das essentielle Hitzeschockprotein 101 (HSP101) nicht exprimiert wird. Um die Rolle von HSP101 für den Leberproteinexport zu klären, wurden transgene HSP101 exprimierende Parasiten erzeugt. Transgene Parasiten weisen in vitro und in vivo schwere Wachstumsstörungen im Leberstadium auf und bieten keinen Impfschutz. Die Ergebnisse legen nahe, dass die Expression von HSP101 streng kontrolliert wird und der Export im frühen Leberstadien nicht wiederhergestellt werden kann. Insgesamt können prä-klinische Studien und die Weiterentwicklung von GAP-basierten Impfstoffkandidaten die laufenden humanen Impfstoffstudien beeinflussen und vorantreiben.
Malaria vaccine candidates providing both safety and efficacy against pre-erythrocytic stages remain largely elusive. Experimental immunizations with live genetically attenuated parasites (GAPs) preventing the development beyond the clinically silent liver stage have proven safe and efficacious. GAP vaccine candidate ΔSLARP, provides the most robust life cycle arrest, however, immunizations do not elicit long-lasting immunity. In contrast, ΔP36p/P36 sporozoites elicit long-lasting immunity, but lead to breakthrough infections during immunizations. This study gives a systematic pre-clinical evaluation of a triple knockout (tKO) GAP by combining ΔSLARP and ΔP36p/P36. Complete arrest of tKO parasites in cultured hepatoma cells and sporozoite-infected mice was confirmed, but time to blood infection after a sporozoite challenge revealed reduced efficacy of the tKO vaccine. While superior immunity can be achieved by a late developmental arrest at liver-to-blood stage conversion, the underlying molecular mechanisms remain elusive. An important question is whether parasite antigens are exposed to the hepatocyte cytoplasm. Protein translocation into the host cell cytoplasm mediated by PTEX, a protein translocon, is absent during liver stage maturation as a core component of PTEX, Heat-shock-protein 101 (HSP101), is not expressed. To clarify the role of HSP101 in liver stage protein export transgenic HSP101 expressing Plasmodium berghei parasites were generated. Parasites expressing elevated levels of HSP101 show severe liver stage growth defects in vitro and in vivo, lack early liver stage export and inferior protection in immunized animals. Our results suggest that HSP101 expression is tightly controlled and PTEX dependent early liver stage export cannot be restored solely by HSP101 overexpression. Overall, pre-clinical analysis and improvement of GAP-based vaccine candidates can inform on-going human vaccine trials and boost malaria vaccine development.

In this study, we determined the immunogenicity of CWP2 and its potential as a vaccine candidate against giardiasis. CWP2 was expressed as a recombinant protein with an hexa-histidine affinity tag and was isolated from inclusion bodies. When BALB/c mice were immunized with CWP2, a specific IgA was detected in the feces. When mice were immunized with CWP2 + cholera toxin, as an adjuvant, IgA in the feces, and IgA, IgG1, and IgG2a in the serum, all specific to CWP2, were detected. Also, CD-1 mice were infected with G. muris and presence of specific IgA antibodies to CWP2 were detected in the feces. This result indicated that CWP2 was recognized by the immune system in a natural infection. IL-4 and IL-5 were released from Peyer's patches (PP) and mesenteric lymph nodes (MLN) cells when stimulated with concanavalin A. In spleen cells, IFN-gamma, IL-4, and IL-5 were released when stimulated with concanavalin A. However, in PP, MLN and spleen cells, the levels of cytokines were barely detectable when stimulated with CWP2. The presence of IgG2a (Th1), IgA and IgG1 (Th2) as the production of IFN-gamma (Th1), IL-4 and IL-5 (Th2) confirmed that CWP2, when presented orally to mice, stimulates both a Th1 and Th2 type immune response, locally and systemically.

Clostridium difficile is the leading cause of hospital acquired diarrhoea. With extended hospitalisation, a high mortality rate and the risk of recurrence, C. difficile infection presents a burden to both patients and the healthcare system. Symptoms of disease are primarily mediated by the two major toxins released by C. difficile and are the focus of current vaccine studies. Little is known of surface proteins in C. difficile, their role in colonisation and their potential as antigens to reduce severity of disease. The surface of C. difficile is composed of a peptidoglycan cell wall and an external S-layer. In many Gram positive bacteria a membrane bound enzyme, sortase, covalently attaches specific proteins to the cell wall. In this study, seven potential sortase substrates were identified and shown to be expressed in C. difficile 630, and at least four were shown to be localised to the cell wall. The substrate CD0183 was shown to need its LPxTG like domain for correct sorting onto the cell wall by sortase, which was shown to be a probable cysteine protease. An efficient cell wall extraction protocol was developed alongside two novel assays for studying the sortase mechanism which could provide future insights into this mechanism of protein sorting. The highly conserved S-layer protein Cwp2 is co-transcribed with CD2790 and cwp66 and its promoter is important for the expression of cwp66, especially during exponential growth. Slayer proteins were shown to potentially reduce colonisation and disease in a multicomponent vaccine, with Cwp2 in particular identified as an antigen which can prevent diarrhoeal symptoms of disease in hamsters. Thus S-layer proteins are potentially important components of a vaccine against C. difficile infection.

A leptospirose é uma doença infecto-contagiosa que acomete os animais domésticos, silvestres e o homem, causada por bactérias do gênero Leptospira. No Brasil, a hemorragia pulmonar constitui o principal fator de risco para o óbito com taxas de letalidade para essa forma da doença superior a 50%. Desta maneira, o desenvolvimento de uma vacina preventiva e o melhor entendimento dos mecanismos de lesão envolvidos nos processos de desenvolvimento da leptospirose nos permitirá propor novas terapêuticas a fim de diminuir a alta letalidade associada à doença. Neste trabalho propusemos investigar doze genes de Leptospira interrogans sorovar Copenhageni. A identificação de uma metaloprotease abre caminho para a investigação de uma possível protease envolvida nos processos hemorrágicos para essa importante zoonose. Esperamos, com os resultados apresentados neste trabalho, contribuir com a caracterização da biologia e patogenicidade da leptospirose.
Leptospirosis is an infectious disease that affects domestic animals, wildlife and humans, caused by bacteria of the genus Leptospira. In Brazil, pulmonary hemorrhage is the major risk factor to death with mortality rates for this form of the disease over than 50%. Thus, the development of a preventive vaccine and better understanding of injury mechanisms involved in leptospirosis would allow us to propose new therapies to reduce the high mortality associated with the disease. In the present study we proposed to investigate twelve genes of Leptospira interrogans serovar Copenhageni. The identification of one protease opens the way for the investigation of a possible protease involved in bleeding processes in this important zoonosis. With the results presented here, we expect to contribute to the biology and pathogenicity characterization of leptospirosis.

A leptospirose é uma doença sistêmica, causada por bactérias patogênicas do gênero Leptospira. O desenvolvimento de novas estratégias para prevenir a doença é necessário. Vacinas surgem como fortes candidatas para contornar o problema. As pesquisas atuais têm interesse em identificar antígenos conservados que estão envolvidos nas interações patógeno-hospedeiro.O presente projeto selecionou três proteínas hipotéticas de L. interrogans para serem caracterizadas quanto ao seu papel na patogênese e avaliadas quanto ao seu potencial protetor. Os genes foram amplificados por PCR e clonados no vetor de expressão PAE. As proteínas recombinantes foram purificadas por cromatografia de afinidade e foram reconhecidas por soro de indivíduos infectados. As proteínas LIC13479 e LIC10050 foram capazes de se ligar a laminina, plasminogênio e fibronectina plasmática. Em relação à LIC10537, dois fragmentos recombinantes foram gerados. Apenas o fragmento 2 foi capaz de interagir com PLG. As proteínas que interagiram com o PLG foram capazes de gerar plasmina As proteínas foram capazes de estimular uma resposta imune e LIC13479 e LIC10050 exerceram proteção parcial no modelo de leptospirose em hamsters.
Leptospirosis is a systemic disease caused by pathogenic bacteria of genus Leptospira. The development of new strategies to prevent the disease is needed. Vaccines emerge as strong candidates to fight the problem.Currently research has focused to identify conserved antigens This project selected three hypothetical proteins of L. interrogans. Thesecoding sequences were characterized for their possible role in pathogenesis and their potential to protect animals against challenge with virulent leptospires. Genes were amplified by PCR and cloned into the expression vector pAE. The recombinant proteins were purified by metal affinity chromatography and were recognized by confirmed human leptospirosis serum samples.LIC13479 and LIC10050 proteins were able to bind with laminin, plasminogen and plasma fibronectin. The coding sequence LIC10537 was cloned in two fragments. Fragment 2was able to interact with plasminogen. All proteins were able to generate active plasmin. The recombinant proteins were able of inducing an immune response. Evaluation of immunoprotection in leptospirosis hamster model followed by challenge with virulent bacteria showed that the recombinant proteins conferred partial protection.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-10T16:21:40Z No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5)
Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-10T16:21:54Z (GMT) No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5)
Made available in DSpace on 2016-05-10T16:21:54Z (GMT). No. of bitstreams: 1 Jessica Mariane Ferreira Mendes Produção... 2016.pdf: 1965926 bytes, checksum: ac5119592b97390470f4914f6fcbddf0 (MD5) Previous issue date: 2016
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
Uma vacina efetiva contra a leishmaniose visceral (LV) canina pode contribuir para o controle da doença no homem. Visando o desenvolvimento de uma vacina contra LV canina, antígenos recombinantes de L. infantum foram selecionados em nosso laboratório pelo uso de uma mistura de soros de seres humanos ou cães naturalmente infectados pela L. infantum. Alguns destes antígenos foram testados em diversos protocolos de imunização, incluindo uso de diferentes adjuvantes, em camundongos ou cães. A imunização de camundongos ou cães com um dos antígenos recombinantes (rLci2B) usado isoladamente ou em associação com saponina induziu resposta imune Th2 ou Th1/Th2, respectivamente, não protetoras contra a infecção experimental. Com a determinação da sequência deduzida de aminoácidos notou-se que a maioria dos antígenos selecionados apresenta um segmento com sequência de aminoácidos única (domínio não repetitivo) e segmentos com sequência de aminoácidos com motivos repetitivos (domínios repetitivos). Possivelmente a incapacidade dos antígenos recombinantes de induzir uma resposta imune predominantemente Th1, protetora contra a LV, seria por conta da presença de domínios repetitivos, que favorecem a apresentação antigênica por linfócitos B e, consequentemente, estimulam uma resposta imune Th2. Para avaliar o direcionamento da resposta imune pelos dois tipos de domínio, novas construções de DNA foram concebidas de modo a codificar apenas domínio(s) não repetitivo(s) ou domínio(s) não repetitivo(s) e domínios repetitivos. OBJETIVOS: Produzir quatro proteínas recombinantes com domínios não repetitivos (rLci2-NT-CT, rLci3-NT-CT, rLci10-NT e rLci12-NT-CT) e avaliar a capacidade desses polipeptídios de induzir resposta imune celular in vitro em cães assintomáticos inoculados por via dérmica com L. infantum. MATERIAIS E MÉTODOS: Foram realizadas: a) a subclonagem de construções de DNA (Lci3-NT-CT, Lci10-NT e Lci12- NT-CT) em um plasmídeo apropriado para expressão em Escherichia coli, b) a determinação de condições apropriadas para produção das proteínas recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R e rLci10- NT) c) a purificação das proteínas recombinantes por cromatografia de afinidade e d) avaliação da capacidade dos polipeptídios de induzir estimulação de células mononucleares sangue periférico (PBMC) de cães assintomáticos inoculados por via dérmica com L. infantum. RESULTADOS: Três (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3- NT-CT, rLci3-NT-2R-CT, rLci10-NT e rLci10-NT-2R) dos quatro pares de polipeptídios recombinantes foram expressos, produzidos e purificados. Três antígenos recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT e rLci3-NT-2R-CT) promoveram a linfoproliferação in vitro utilizando PBMC de cães assintomáticos inoculados por via dérmica com L. infantum CONCLUSÕES: Três das seis proteínas produzidas induziram a linfoproliferação, sendo a maior linfoproliferação encontrada para PBMC estimulado com a proteína sem domínios repetitivos (rLci2-NT-CT). Avaliações adicionais são necessárias para comprovar a utilidade destas moléculas em formulação de vacina contra leishmaniose visceral canina.
An effective vaccine against visceral leishmaniasis (VL) dog can help to control the disease in man. Aiming at development of a vaccine against canine VL, recombinant antigens of L. infantum were selected in our laboratory by using a mixture of sera from humans or dogs naturally infected with L. infantum. Some of these antigens were tested in different immunization protocols, including use of different adjuvants in mice or dogs. The immunization of mice or dogs with a recombinant antigens (rLci2B) used alone or in combination with saponin induced Th2 response or Th1 / Th2, respectively, did not protective against experimental infection. With the determination of the deduced amino acid sequence it was noted that most of the antigens selected segment has a unique amino acid sequence (non-repetitive domain) and segments of amino acid sequence with repetitive motifs (repetitive domains). Possibly the inability of recombinant antigens to induce an immune response predominantly Th1 protective against LV would be due to the presence of repetitive domains that promote antigen presentation by B cells and thus stimulate an immune response Th2. To assess the direction of the immune response by two types of domain, new DNA constructs were designed to encode only the domain (s) not repetitive (s) or domain (s) not repetitive (s) and repetitive fields. MATERIALS AND METHODS: Were performed: a) subcloning DNA constructs (rLci3-NT-CT, rLci10-NT and rLci12-NT-CT) into a suitable plasmid for expression in Escherichia coli, b) determining the appropriate conditions for the production of proteins recombinant (rLci2- NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R and rLci10-NT) c) purification of recombinant proteins by chromatography affinity d) evaluating the ability of polypeptides rLci2-NT-CT, rLci3-NT-CT and rLci10-NT to induce stimulation of peripheral blood mononuclear cells (PBMC) from healthy dogs inoculated dermal with L. infantum. RESULTS: Three (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3-NT-CT, rLci3-NT-2R-CT, rLci10- NT and rLci10-NT-2R) of the four pairs of recombinant polypeptides are expressed, produced and purified. Three recombinant antigens (rLci2-NT-5R-CT, rLci2-NT-CT and rLci3-NT-2R-CT) promoted lymphocyte proliferation in vitro using asymptomatic dogs PBMC inoculated dermal L. infantum CONCLUSIONS: Three of the six proteins produced induced lymphoproliferation, most lymphoproliferation was found to PBMC stimulated with the protein without repetitive domains (rLci2-NT-CT). Additional evaluations are necessary to confirm the utility of these molecules against canine visceral leishmaniasis vaccine formulation

A esquistossomose é uma doença causada por trematódeos do gênero Schistosoma. Dentre os genes identificados no transcriptoma do parasita, membros da família gênica SmVAL (Schistosoma mansoni Venom Allergen-Like) foram apontados como candidatos vacinais. SmVALs foram identificadas em secreções de cercárias e esquistossômulos cultivados in vitro, os transcritos SmVAL4 e 24 foram localizados nas glândulas acetabulares de germ ball e a proteína nativa SmVAL4 foi identificada em extrato de cercárias, indicando funções durante a penetração da pele. Já os transcritos SmVAL13 e 14 foram localizados na glândula esofágica anterior de vermes adultos, sugerindo papéis no processo de alimentação sanguínea. A imunização com as proteínas rSmVAL4, 6, 7, 13, 14 e 18 coadministradas não protegeu camundongos contra o desafio experimental, porém, observou-se uma diminuição do número de fêmeas e do número de ovos no grupo imunizado. A investigação de funções para as proteínas secretadas mostrou que a rSmVAL18 interage com plasminogênio in vitro favorecendo a invasão do hospedeiro.
Schistosomiasis is a disease caused by trematodes of the genus Schistosoma. Among the genes identified in the parasite transcriptome, members of SmVAL (Schistosoma mansoni Venom Allergen-Like) gene family were proposed as vaccine candidates. SmVALs were identified in cercariae and schistosomule secretions in vitro, the SmVAL4 and 24 transcripts were located to the germ ball acetabular glands and SmVAL4 native protein was identified in cercariae extract, indicating functions in skin penetration. On the other hand, SmVAL13 and 14 transcripts were located to the anterior esophageal gland of adult worms, suggesting roles in the blood feeding processes. Immunization with rSmVAL4, 6, 7, 13, 14 and 18 proteins co-administered did not protected mice against experimental challenge, however, there was a decrease in the number of females and the number of eggs in the immunized group. The investigation of functions for secreted proteins showed that rSmVAL18 interacts with plasminogen in vitro thus favoring the host invasion.

Streptococcus pneumoniae é o agente causador de diversas doenças, tais como meningite e pneumonia. PspC (Pneumococcal surface protein C) foi descrito como um importante candidato vacinal proteico de ampla cobertura e com baixo custo de produção. Trata-se de um fator de virulência, capaz de ligar-se ao Fator H (FH) e a IgA secretório (sIgA). Como PspC é um antígeno polimórfico, é crucial a avaliação da sua variabilidade. Foi determinado o grupo de PspC de treze linhagens de pneumococo isoladas no Hospital Universitário da Universidade de São Paulo. Soros contra diferentes grupos de PspC foram produzidos e PspC do grupo 3 (PspC3) foi capaz de induzir anticorpos que reconhecem diferentes grupos de PspC. Foi observada ainda uma pequena redução na ligação de FH e sIgA por anticorpos anti-PspC3 em ensaios in vitro. No entanto, não foi possível observar proteção contra um modelo de colonização da nasofaringe de camundongos através da imunização com PspC3, possivelmente por deficiências no modelo experimental.
Streptococcus pneumoniae is the causative agent of several diseases, such as meningitis and pneumonia. PspC (Pneumococcal surface protein C) has been described as an important vaccine candidate protein as it could provide wide coverage with low cost of production. PspC is a virulence factor capable of binding to Factor H (FH) and secretory IgA (sIgA). PspC is polymorphic antigen, and therefore it is crucial to evaluate its variability. In the present work we have determined the PspC group of 13 pneumococcal isolates obtained at the University Hospital of the University of São Paulo. Antisera against different PspC groups were produced and PspC group 3 (PspC3) was able to induce antibodies that recognized different groups of PspC. Antibodies to PspC3 reduced binding of FH and sIgA to pneumococcus in in vitro assays. However, no protection was observed against a murine model of nasopharyngeal colonization by immunization with PspC3. This was possibly due to deficiencies in the experimental model.

Neisseria gonorrhoeae is a strict human pathogen and the causative agent of the sexually transmitted disease, gonorrhea. Gonococcal (GC) diseases remain one of the most reported sexually transmitted infections (STI) worldwide, representing a significant threat to reproductive health and burden on global health systems, accounting for 541,987 disability adjusted-life years in the year 2011. Infection by N. gonorrhoeae also increases the likelihood of patient acquisition and transmission of human immunodeficiency virus (HIV). Unfortunately, antibiotic treatment to gonococcal diseases is being threatened by the rapid spread of resistance to third-generation cephalosporins, the remaining treatment option used in clinics. The urgency of the situation is compounded by the relative lack of immunological protection conferred by previous infection by the bacterium. In response to the emergence of multidrug resistance, renewed energies are being directed towards the development of an effective, broadly protective vaccine. Difficulties in vaccine development arise from a lack of known correlates of protective immunity; there is no known broadly cross-protective immunity to GC and a truly reflective animal model has not been available. Nonetheless, previous studies have indicated that porins, neisserial major outer membrane proteins, are promising vaccine candidates. PorB makes up over 60% of the bacterium's outer membrane content and is involved in solute and ion exchange, invasion of target host cells, and evasion of host immunity. Porins from both the gonococcus and the meningococcus have been shown to have immunostimulatory activity, boosting B and dendritic (DC) cell proliferation and maturation in the absence of an exogenous adjuvant as mediated by TLR2 and MyD88. Importantly, as a potential vaccine candidate, PorB has relatively low antigenic variability, and can induce bactericidal antibodies. Gonococcal PorB was purified from a genetically modified strain, MS11delP3, which lacks another outer membrane protein, RMP, which is known to induce bactericidal blocking antibodies. PorB was formed into pure protein micelles, termed proteosomes, to protect the integrity of the native trimeric structure. Our study demonstrated that gonococcal PorB is able to stimulate both human embryonic kidney (HEK) cell line that overexpresses TLR2 and mouse primary macrophages (similar to the meningococcal PorB). To test PorB's immunogenicity, mice were immunized three times at two week intervals with PorB and porin specific IgG levels were measured. Unfortunately, PorB elicited lower levels of porin specific IgG than what was expected, which may be due to technical issues. We are currently investigating various possibilities. In addition, further immunization studies shall be carried out to better contextualize these results.

abstract: CTB-MPR649-684 is a translational fusion protein consisting of the cholera toxin B subunit (CTB) and the conserved residues 649-684 of gp41 membrane proximal region (MPR). It is a candidate vaccine component aimed at early steps of the HIV-1 infection by blocking viral mucosal transmission. Bacterially produced CTB-MPR was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice and rabbits. However, the induction of high-titer MPR specific antibodies with HIV-1 transcytosis blocking ability remains a challenge as the immuno-dominance of CTB overshadows the response to MPR. X-ray crystallography was used to investigate the structure of CTB-MPR with the goal of identifying potential solutions to improve the immune response of MPR. Various CTB-MPR variants were designed using different linkers connecting the two fusion proteins. The procedures for over-expression E. coli and purification have been optimized for each of the variants of CTB-MPR. The purity and oligomeric homogeneity of the fusion protein was demonstrated by electrophoresis, size-exclusion chromatography, dynamic light scattering, and immuno-blot analysis. Crystallization conditions for macroscopic and micro/nano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro/nano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2015

In the second part of the project, the immunology of PfMSP-133 was studied. During the invasion of merozoites, PfMSP--142 is processed into two fragments with molecular weight of 33kDa and 19kDa. The 19kDa fragment (PfMSP-119) originating from the carboxyl--terminal of PfMSP--142 is relatively more immuno-dominant in different malarial species such as P. falciparum, P. vivax and P. yoelii. In the past, only limited researches about PfMSP-1 33 were performed. Apart from its difficulty in expression, PfMSP-1 33 was also believed to be incapable of inducing protection.
Nevertheless, following the breakthrough of expressing recombinant PfMSP-1 33 in our laboratory, we have demonstrated in this study that recombinant MSP-133 can elicit antibodies with a titer up to a million. Also, we observed that MSP-133 can help MSP-119 to induce protective immunity and such effect is independent from the covalent linkage between these two proteins. Most importantly, our results show that recombinant PfMSP-133 can elicit the production of antibodies that can potentiate the inhibitory effect of anti-MSP-142 serum at high serum dilution. Results of this study give new insights in malarial vaccine development in terms of optimizing the use of adjuvant and immunization regimens.
The 42kDa carboxyl terminal fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP--142) is one of the most promising candidate antigens in the development of malarial vaccine. In vivo experiments in the 1990's showed that Aotus monkeys immunized with PfMSP--142 were protected from malarial challenge. Later on, other experiments also demonstrated the possibility of using recombinant PfMSP-142 as candidate antigen for malarial vaccine. Previously, recombinant PfMSP-142 (Bvp42) was expressed with the baculovirus expression system and characterized in our laboratory.
The aim of the first part of this project is to improve the production of Bvp42. Experimental results have shown that the expression level of Bvp42 was increased under a BMN compatible baculovirus expression vector---pVL1393. Besides, a codon optimized MSP-142 nucleotide is constructed for the construction of a baculovirus carrying codon optimized MSP-142 gene and aimed for higher expression level. Unfortunately, no Bvp42 expression is observed in the transfection samples and the reason of this observation is unclear. Meanwhile, the purification of Bvp42 was also improved. Pretreatment of the hemolymph with Q--sepharose before affinity chromatography could enhance the purity of the final product.
Yuen, Sai-hang Don.
"July 2007."
Adviser: Walter K. K. Ho.
Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0220.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 183-195).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

Our overall research goal is focused on the development of a malaria transmission-blocking vaccine (TBV). The antigenic target, Pfs48/45 protein, is expressed on Plasmodium gametocytes, which are stages responsible for establishing parasite infection in the mosquito vector. The epitopes recognized by functional antibodies targeting Pfs48/45 are disulfide-bond (S-S) constrained, conformational epitopes. As Pfs48/45 protein has not been crystallized, precise location of the S-S bonds and the topology of epitopes are unknown. It has been shown previously that the ability to reduce S-S in antigens can greatly influence the epitopes presented by antigen-presenting cells (APCs) and thus influence induction of effective immune responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is an enzyme expressed in APCs that mediates reduction of S-S bonds contained within antigens, for subsequent display of peptides on MHC molecules. Using non-reduced (NR) and reduced/alkylated (RA) Pfs48/45 antigens, we sought to investigate the role of GILT on induction of protective immunity. We hypothesized that the ability to reduce S-S bonds in Pfs48/45 will impact the generation of T cell epitopes, and thus influence helper T cell responses required for B cell stimulation and production of protective antibody. We conducted immunogenicity studies in wild type (WT) and GILT-/- (KO) mice using the two structural forms of Pfs48/45 and analyzed immune responses to full length Pfs48/45, five overlapping fragments and 39 overlapping peptides. Results indicated that generation of Pfs48/45 antibodies is not significantly impacted by the availability of GILT, however there was uniquely Th2-biased T and B cell responses in the KO mice, and a contrasting Th1 bias in WT mice. Results also revealed possible effects of GILT on induction of long-lived plasma cells and memory B cells responsible for resting and antigen-recall responses to Pfs48/45. Data presented also shows reduced immunogenicity of the RA Pfs48/45 antigen and immune responses differed in magnitude and specificity between male and female animals. Overall, we aimed to gain a better understanding of the immunological mechanisms critical to generate protective and lasting immunity against Pfs48/45. These and future studies will contribute significantly to our understanding of antigenic features of Pfs48/45 important for use as a TBV.
acase@tulane.edu

Miu Fei Fei.
On t.p. "42" are subscripts following the word "MSP-1" in the title.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (leaves 117-125).
Abstracts in English and Chinese.
ACKNOWLEGEMENTS --- p.i
ABSTRACT --- p.ii
TABLE OF CONTENTS --- p.v
LIST OF FIGURE --- p.vii
LIST OF ABBREVIATIONS --- p.ix
CHAPTERS:
Chapter 1. --- BACKGROUND OF MALARIA
Chapter 1.1 --- Epidemilogy --- p.2
Chapter 1.2 --- Mode of Infection --- p.4
Chapter 1.3 --- Conventional Control & Vaccination --- p.9
Chapter 1.4 --- Vaccine Candidate PfMSV-142 --- p.16
Chapter 1.5 --- Cloning and Expression of pfMSP-142 --- p.26
Chapter 1.6 --- Aims of Study --- p.32
Chapter 2. --- Materials and Methods
Chapter 2.1 --- Materials --- p.32
Chapter 2.2 --- Methods --- p.38
Chapter 3. --- Construction of recombinants N-PfMSP-142 and C- PfMSP-142
Chapter 3.1 --- Construction of C-PfMSP-l42 --- p.51
Chapter 3.2 --- Construction ofN-PfMSP-l42 --- p.56
Chapter 4. --- Purification with IMAC
Chapter 4.1 --- Immobilized Metal Affinity Chromatography (IMAC) --- p.58
Chapter 4.2 --- Purification ofN-PfMSP-l42 --- p.61
Chapter 4.3 --- Purification profile of N-PfMSP-142 --- p.68
Chapter 4.4 --- Purification of C-PfMSP-l42 --- p.70
Chapter 4.5 --- Purification profile of C-PfMSP-142 --- p.73
Chapter 4.6 --- Expression pattern of recombinants PfMSP-142 --- p.76
Chapter 5. --- Purification combined with other chromatography method
Chapter 5.1 --- Affinity chromatography --- p.78
Chapter 5.2 --- Gel filtration chromatography --- p.80
Chapter 5.3 --- Ion exchange chromatography --- p.83
Chapter 5.4 --- Conclusion --- p.93
Chapter 6. --- Characteristic of IMAC products --- p.94
Chapter 7. --- Characteristic of N-hisPfMSP-l42 & C-hisPfMSP-l --- p.42
Chapter 7.1 --- Immunogenitcity of N-PfMSP-l42 and C-PfMSP-142 --- p.100
Chapter 7.2 --- Competitive ELISA --- p.105
Chapter 8. --- Discussion --- p.107
REFERENCE --- p.117

Members of the family Chlamydiae cause a wide range of diseases. Chlamydia trachomatis and C. pneumoniae are most commonly associated with human disease. C. psittaci and C. pecorum are largely animal pathogens, although C. psittaci can cause pneumonia in the elderly and immunocompromised. A vaccine against these pathogens is desirable, but although multiple vaccine regimens have been examined, none have proven truly effective. Studies were conducted to evaluate the use of recombinant vaccinia virus (VV) vectors encoding chlamydial proteins as vaccine candidates using a guinea pig model. In the first study, guinea pigs were immunized with varying amounts of attenuated VV encoding either the M6 protein of Streptococcus pyogenes or chloramphenicol acetyl transferase (CAT) from E. coli. The purpose of this study was: (1) determine how much attenuated virus can be given intranasally to guinea pigs without causing death; (2) characterize the humoral and secretory antibody response to both the viral vector and M6 protein; and (3) develop a protocol for animal manipulation, and sample collection and storage for use in future research. The results obtained indicate that 10⁹ PFU of attenuated VV can be given intranasally to guinea pigs. Serum IgG was detected against VV proteins, as determined by immunoblotting. Antibodies against M6 could not be similarly detected in serum, or by direct enzyme linked immunosorbant assay (ELISA). IgG could not be detected by immunoblotting against either VV or M6 in saliva. The purpose of the second part of this research was to evaluate the potential efficacy of a vaccine using the C. psittaci guinea pig inclusion conjunctivitis (GPIC) strain proteins IncA and TroA. Guinea pigs were immunized intranasally with either PBS, control vaccinia virus, rVV:IncA, or rVV:TroA. Three weeks after immunization animals were challenged by ocular infection with C. psittaci elementary bodies (EBs). Eye swabs were taken following challenge and titered to determine the chlamydial load. Results indicate that rVV:TroA provides no protection against chlamydial challenge. Titers from rVV:IncA immunized animals appeared to be somewhat lower than those of the controls on day 4 post-challenge. This difference, however, proved not to be statistically significant. A single immunization with rVV:IncA or rVV:TroA was thus shown not lead to a protective immune response in guinea pigs under the conditions tested.
Graduation date: 2002

碩士
國立臺灣大學
口腔生物科學研究所
94
Enterovirus 71 has been the most important enterovirus to cause hand-foot-and-mouth disease accompanied with neurological complication. EV71, like the poliovirus, belongs to the Picornaviridae family and there are four kinds of structural proteins, VP1-VP4, to assemble the virions. It is generally considered that VP1 is the most important antigenic determinant of EV71. Therefore, previous studies on the EV71 vaccine most focused on the inactivated virus and VP1, but inactivated virus vaccine had better protection than VP1 subunit vaccine. In addition, studies on poliovirus showed that three to four neutralizing antigenic sites have been described, involving residues of all three major structural proteins, VP1-VP3. Based on the reasons above, we want to identify neutralizing epitopes of EV71 Caspid, other than VP1, to improve vaccine. We purified other recombinant caspid proteins, other than VP1, to immunize BALB/c mice accompanied with CFA/IFA or cholera toxin as adjuvant. We demonstrated the VP0 (VP0 is the propeptide of VP2 and VP4) and VP3 can induce specific antibodies. In the neutralization test, we also found that anti-VP0 and anti-VP3 serum can protect RD cell against virus infection as well as anti-VP1 serum. Further, serum samples from EV71-immunized mice had the best neutralization effect. About the antigen saturation study, only inactivated virus could block neutralization effect of anti-virus serum. We suggested that denatured VP1, VP3, and VP0 might lose most of conformational epitopes during the purification and had low affinity with anti-virus serum. In the other hand, we estimated the component of anti-virus serum by ELISA and we found that VP1-specific antibody was more than VP3 and VP4-specific antibody. Then, we combined different structural proteins with different ratio to immunize BALB/c mice via nasal route for the reason that induction of mucosal immunity to protect virus infection in the first line of defense. The results showed that only inactivated virus can elicit great magnitude of immune response. In conclusion, antibodies induced by EV71 capsid proteins, other than VP1, had neutralization effect and whole virus could induce the best neutralizing antibodies and mucosal immunity. We estimated components of anti-virus serum by ELISA and considered that VP1 might be more important neutralizing epitopes in virus infection. We considered that intact virus is the proper candidate of the EV71 vaccine development. Thus, we will design enterovirus-like particle for development of mucosal vaccine.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique.
Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.

Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

碩士
國立中興大學
微生物暨公共衛生學研究所
106
Chicken anemia virus（CAV）is one of the important pathogens affecting poultry industry globally. The virus causes mortality, production losses, immunosuppression, sensitive to infections and vaccination failures in infected chickens. Currently, only live attenuated chicken infectious anemia（CIA）vaccines are available for administering in breeders older than 8 weeks that induce maternal antibodies to protect their progeny till 3 weeks. However, the vaccine viruses could induce vertical and horizontal infection to other chickens. These disadvantages of the traditional vaccines induce the idea to develop novel CIA vaccines. According to previous studies, VP1 is the only structural protein forming the viral capsid, and the correct folding of VP1 requires expression of VP1 and VP2 in the same cell. Interleukin-12（IL-12）is a proinflammatory cytokine, which is responsible for enhancing cytotoxicity of NK cells and T cells. Moreover, IL-12 facilitates the generation of related TH1 cytokines, and promotes the production of immunoglobulins by stimulating helper T cells. Therefore, IL-12 can be serve as a biological adjuvant. In this study, the baculovirus expression system was used to produce the structural protein of CAV and chicken IL-12（chIL-12）for developing a subunit vaccine candidate. The VP1 and VP2 of CAV and the chIL-12 gene codons were optimized for expressing in the insect cells, and the recombinant proteins were expressed by the baculovirus expression system. Western blot and indirect immunofluorescence assays were used to verify the expression of recombinant proteins. The results showed that VP1 and chIL-12 can be expressed in eukaryotic cells. We found that VP1 predominantly aggregated in the nucleus, and the amount of expression level needs to be further optimized. Biological adjuvant property of chIL-12 has been tested by inducing high expression level of IFN-γ in the recombinant chIL-12 stimulated splenocytes. Finally, different concentrations of recombinant VP1 and chIL-12 has been combined together to assess immune responses in chickens, and the results showed that VP1 combined with chIL-12 achieved the highest titers of CAV specific antibody than those of other groups. Taken together, the recombinant VP1 could be a candidate of CAV subunit vaccine, and co-immunization of chIL-12 can induce a strong immune response in vaccinated chickens.

Carbohydrates and proteins represent two large groups of biomolecules which are tremendously important for biological processes in health and disease state. Although protein-structures are encoded in the genome, cellular glycan structures are template independent and can only be addressed in an indirect manner. The development of metabolic oligosaccharide engineering (MOE) gave rise to new methods to study carbohydrate structures in the context of different disease settings and in different organisms. While in many cases mannose derivatives are used to study the sialic acid structures in cancer cells, this work presents the results on the metabolic incorporation of galactose derivatives into cell membrane glycans of human hepatic cells. Three unnatural galactose derivatives containing terminal alkene groups in C2 or C6 position were synthesized and their reaction rates in inverse electron demand Diels Alder reactions (iEDDA) were evaluated, by using a high-throughput screening method in 96-well plates. It was shown that none of the developed galactose derivatives exhibit any cell toxic effect in HepG2 or Huh7 cell lines. Furthermore, all monosaccharides could be successfully incorporated in cell membrane glycan structures of both cell lines and the localization on the cell membrane was confirmed by co-localization with a plasma membrane dye. After developing this incorporation and labeling strategy of unnatural galactose derivatives in the cell membrane of human hepatic cells, the change in incorporation during an infection of these cells with Plasmodium berghei sporozoites was investigated. By using different techniques, such as confocal microscopy, flow cytometry and imaging flow cytometry, only a small trend for an increased uptake of the unnatural galactose derivative in P. berghei infected cells was observed. To explain this result, the pathway for the diffusion of the unnatural galactose derivative was determined. The application of specific and non-specific inhibitors for the glucose transporter GLUT1 revealed that this transporter is involved in the delivery of galactose derivatives into cultured cells. The enhanced translocation of this transporter to the surface of infected hepatic cells explains the observed tendency for an increased incorporation of the unnatural galactose derivative in these cells. Apart from cell studies, MOE was applied for the first time to study a possible transfer of galactose monosaccharides from the mosquito host to the parasite. Biosynthetic pathways for glycan assembly in the parasite are poorly understood. Suggestions on the participation of the mosquito host in some of these pathways, led to the idea to apply MOE in this situation. It was possible to show an uptake of the presented galactose derivatives by the mosquito but only reduced transfer to the parasite seems to occur. In addition to the development of monofunctional galactose derivatives, also a bifunctional derivative containing two orthogonal reporter groups was synthesized. However, so far it was not possible to achieve a metabolic incorporation or labeling of this derivative on cell membrane glycans. After developing cellular tools to study carbohydrate structures, a site-selective method for protein modification was generated, to be used for the development of new glycoconjugate vaccine candidates. By introducing selectively two dehydroalanine residues in place of the disulfide bond C186-C201 of the immunogenic protein CRM197, a new chemical moiety for the conjugation of carbohydrate antigens was obtained. It was shown that these moieties can be used for the selective introduction of polysaccharide antigens from group B Streptococcus (GBS) or Streptococcus pneumoniae. Both types of glycoconjugates could be synthesized and first trials on the purification methods were undertaken. This concept will be developed further for future vaccine candidates. Finally, a synthetic method was developed which could facilitate the synthesis of defined antigenic oligosaccharide structures. This method uses the thiophilic promoter O-mesitylenesulfonylhydroxylamine (MSH) for the activation of thioglycoside donors. It was demonstrated that different thioglycoside donors are activated with different kinetics, depending on the presented protecting groups or the anomeric leaving group. Apart from applying the developed activation method for the synthesis of several glycosylation products, the sequential activation of S-alkyl before S-phenyl anomeric groups was shown during the synthesis of a model trisaccharide. Overall, bio-orthogonal methods were developed and applied for the investigation of carbohydrate structures in the context of malaria disease, and for the site-selective modification of protein carriers during the development of glycoconjugate vaccine candidates.