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No description available.

No description available.

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.

Streptococcus pneumoniae is one of the leading causes of morbidity and mortality in children under 5. Low-income countries are disproportionally affected and data in these settings are lacking. Effective strategies to control disease include infant immunisation with pneumococcal protein-polysaccharide conjugate vaccines. However, ongoing surveillance of carriage and disease are important to understand the impact of vaccination within communities. This thesis evaluated nasopharyngeal (NP) carriage in 3 generations, following introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the UK. NP carriage was also compared between rural and urban Nepalese children and a novel method of delayed culture and transport was assessed. Finally, the immunogenicity of a 10-valent pneumococcal conjugate vaccine administered in a 2-dose priming schedule without a booster was compared to a 3-dose priming schedule with a booster in Nepalese infants. Key findings include carriage rates in UK children being similar to pre-PCV7 (7 valent pneumococcal conjugate vaccine) carriage rates at 47% with low carriage rates in seen in adults. PCV7 serotypes accounted for 1.5% of carriage isolates in children, 0% in parents and 15.4% in older adults. In Nepalese children carriage was higher in a rural (69.2%) compared to an urban setting (40.9%) and delayed culture and transport using silica desiccant packets (SDP) provided a reliable, albeit underestimated, estimate of carriage. Finally this author demonstrated that following primary immunisation and boosting, there was no difference in immune responses to serotypes 1, 5 and 14 with a 2 dose priming schedule compared to a 3-dose schedule. At 2-4 years of age a significantly higher proportion of vaccinees in the 2+1 group had ≥0·2µg/mL IgG for serotypes 1, 5, 6B and 18C compared to vaccinees in the 3+0 group.

At the author’s request, the abstract has been removed due to the confidential nature of the thesis. It will be added once the embargo period has passed.

Neisseria meningitidis (Nm) disease occurs worldwide. Disease incidence rates can vary from 1 to 1000 cases per 100,000 with the highest incidence found in the sub-Saharan Africa meningitis belt. Nm has evolved a number of mechanisms to evade host immunity. This includes the production of genetic variants through re-combinatorial events, which is thought to have contributed to the evolution of hyper-invasive lineages that are largely responsible for meningococcal disease. Antigenic diversity of Nm surface proteins has been the main limitation in the design of broadly protective vaccines, particularly against capsular serogroup B strains. To overcome this problem, several Nm genomes have been sequenced in an effort to find highly sequence-conserved surface antigens recognized by the human immune system in order to develop a vaccine, which would be broadly protective against disease. Nm genomes contain over 2 million base pairs that contain between 2000 and 2500 open reading frames. Add to this the difficulty of identifying highly conserved recombinant antigens with strong intrinsic immunostimulatory properties, makes vaccine design and development a daunting task. Recent advances in our understanding of the interactions between innate and acquired immunity, and the discovery of pattern recognition receptors, including Toll-like receptors (TLRs), have ushered in a new set of adjuvant compounds, TLR agonists, which invoke strong humoral and cellular responses with nominal toxicity and adverse reactions. These insights have opened up new areas of vaccine research to combat invasive Nm disease.

Soil-transmitted helminths are a major cause of morbidity for humans and their livestock. A combination of better sanitation, anthelminthic drugs and vaccines are predicted to reduce the morbidity of these parasites in humans. The drugs currently used to treat these infections, albendazole and mebendazole, are fairly ineffective against Trichuris trichiura (human whipworm), and there are reports of drug resistance arising within parasite populations in Vietnam and Zanzibar. There are also no commercially available vaccines against human STH species, and very few against their veterinary counterparts. The murine whipworm, T. muris, has been used for over 50 years as a model for T. trichiura. These parasites share homology at the genomic and transcriptomic levels, and the immune responses associated with both acute and chronic infection have been well studied using the T. muris mouse model. T. muris excretory/secretory products have been studied in the context of vaccination for over four decades, however relatively little progress has been made towards identifying the molecular components that stimulate protective immunity following vaccination or during acute infection. Here, a stringent selection protocol was developed using chromatography and mass spectrometry methods combined with a measurement of T cell cytokine production. The work presented in this thesis provides a novel framework for identifying potential immunogenic candidates within adult T.muris excretory/secretory products. Exosome-like vesicles isolated from adult T. muris ES were also explored as a source of host protective material. Vaccination with exosome-like vesicles protected male C57BL/6 mice from a subsequent low dose infection, which would ordinarily progress to chronicity, and a number of potential immunogenic candidates were identified. Over the course of this thesis, several important observations were made relating to characteristics of the immune response induced by vaccination with ES. Firstly, proteinaceous material is likely to be responsible for the host protective properties of ES. Secondly, vaccination with ES products stimulates long-lasting immunity. Thirdly, vaccination with ES collected from both larval and adult stages stimulates protective immunity. The number of potential immunogenic candidates has also been narrowed down from over four hundred to just eleven. Given the homology between T. muris and T. trichiura at both the genomic and transcriptomic levels, this work has the potential to advance vaccine design for T. trichiura and other Trichuris parasites.

acase@tulane.edu / 1 / Taylor Foreman

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis. / Nicole L Kikendall