No description available.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

Thesis (PH.D.)--University of Wollongong, 2004. / Typescript. Bibliographical references: leaf 231-250.

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: RTS,S candidate malaria vaccine has been shown to be highly immunogenic in children and infants, but the protective immune mechanisms still remain to be clearly elucidated. It is believed that RTS,S elicits a strong neutralizing humoral immune response directed against surface-exposed sporozoite proteins and cell mediated immune (CMI) responses characterized by predominantly CD4+ Th1 cells. The objective of this study was to investigate humoral and cell-mediated immune responses to the RTS,S/AS02D malaria vaccine and its association with protection against infection and disease by P. falciparum. Methodology and Principal Findings: This secondary data analysis from data of a phase I/IIb randomized, double-blind, controlled trial, included 154 healthy infants living in rural Mozambique, previously immunized with RTS,S/AS02D candidate malaria vaccine or the control Engerix-B™ vaccine. Antibodies against circumsporozoite protein (CSP) and hepatitis-B surface antigen (HBsAg) were measured with a standard ELISA. Fresh blood intracellular staining assay was performed to evaluate the expression of IL-2 and IFN-γ by CD4+ and CD8+ cells in response to in vitro stimulation of specific peptides. Data was evaluated for association with the risk of malaria detected by both active and passive case detection of infection over a period of 6 months post dose 3. Anti-HBs antibody geometric mean titers declined from 10,082 mIU/mL one month post Dose 3 to 2,751 mIU/mL at 12 months post Dose 3 in the RTS,S/AS02D group; anti-HBs v geometric mean titers were 392.4 mIU/mL and 263.9 mIU/mL, respectively in the Engerix- BTM group. Anti-CSP antibody geometric mean titers declined from 199.9 EU/mL one month post Dose 3 to 7.3 EU/mL at 12 months post Dose 3 in the RTS,S/AS02D group. Median stimulation indices of HBs-specific IL-2 and IFN-γ producing CD8+ T cells was higher in the RTS,S/AS02D group than in control group (Wilcoxon rank sum p-values for IFN-γ = 0.015, for IL-2 = 0.030) at 10.5 weeks post immunization. Median stimulation indices of anti-CSP specific IFN-γ producing CD8+ T cells at the same time point was 1.13 (IQR: 0.79 - 1.67; p=0.029). For specific IL-2-producing CD4+ T cells, the median SI was 1.14 (IQR: 0.74 – 1.60, p=0.043) at 10.5 weeks post dose three. The reduction in hazards of malaria infection were 18.3 % (95% CI: -267.9 – 81.8, p=0.793) and -12.0 % (95% CI: -295 – 68.2, p=0.86) for specific IL-2 CD4+ stimulation indices; For specific CD8+ IFN-γ stimulation indices the hazards were -103.6% (95% CI: -690.9 – 47.6; p=0.305) and 48.8% (95% CI: -97.0 – 86.7; p=0.33) at four and 10.5 weeks post immunization respectively. Conclusion: The RTS,S/AS02D vaccine was immunogenic and has elicited detectable levels of CSP specific cell mediated responses. No evidence of association was found between the antibodies anti-CSP and specific cell mediated responses and the risk of malaria.

Abstract not available

Malaria is a life-threatening parasitic disease endemic throughout the world. Control methods for malaria are becoming less reliable; thus, efforts to develop a safe and effective vaccine are critical. Immunity to malaria requires both cell- and humoral-mediated immunity, CMI and HMI, respectively. CD4+ T cells play a central role in protection against blood stage Plasmodium infection. Given that clinical features of malaria are caused by blood stages, a vaccine against this stage will be very effective in reducing morbidity and mortality. During the blood stage, purine nucleotides, which are essential for parasites’ survival and proliferation, are in high demand. The inability of the parasite to engage in de novo synthesis of purine nucleotides makes the enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) an essential nutrient salvage enzyme. HGXPRT is located in electron-dense regions in merozoites and in vesicles in the red cell cytoplasm. In contrast to other blood stage antigens, those located on the merozoite surface are targets of HMI. To advance HGXPRT as a malaria vaccine candidate, fermentation and purification of the protein from Plasmodium falciparum (PfHGXPRT) was performed using facilities at Q-Gen, the Queensland Institute of Medical Research (QIMR). Escherichia coli carrying PfHGXPRT gene were a gift in-kind from the University of Queensland (UQ). Recombinant PfHGXPRT expressed in E.coli was purified using anion exchange liquid chromatography and gel filtration techniques. Three methods were used to confirm the Q-Gen PfHGXPRT identity: (1) Western blotting showing identical bands of UQ PfHGXPRT and Q-Gen PfHGXPRT at 26 kDa; (2) N terminal sequencing was compatible with the PfHGXPRT sequence; and (3) mass spectrometry showed homogeneity by giving a subunit molecular mass of 26,231 Da. The purification method used is reproducible and affordable, yielding reasonably pure protein for animal experimentation. Following purification of PfHGXPRT, its efficacy as a subunit vaccine candidate in a rodent model of infection was examined. Multiple rodent models of malaria infection were assessed and it was determined that Plasmodium chabaudi AS (P. chabaudi AS) exhibited the highest cross-reactivity against PfHGXPRT in mice. Hence, P. chabaudi AS was chosen as the appropriate rodent model for study in this thesis. Natural immunity against PfHGXPRT during a blood stage P. chabaudi AS infection was assessed by testing sera and splenocyte responses to PfHGXPRT. IFN- and IL-4, as well as antibodies specific for PfHGXPRT, could be detected after infection, suggesting that PfHGXPRT is a target of natural immunity during the blood stage infection. Therefore, further studies of protective immunity generated by immunisation with PfHGXPRT were conducted, specifically to determine their protective efficacy and to determine immune mechanisms elicited by immunisation. Mice immunised with PfHGXPRT and challenged with P. chabaudi AS developed a slightly reduced parasitaemia. T-cell proliferation, but not antibody responses, was detected after immunisation. Protective mechanism(s) were assessed by adoptively transferring immune CD4+ T cells, B cells or sera to naïve SCID mice followed by parasite challenge. Only recipients of immune CD4+ T cells showed extended survival. Nevertheless, immunisation with PfHGXPRT followed by sub-patent infection induced better protection than immunisation with PfHGXPRT alone, which appeared to be related to CD4+ T cells. Reduction of parasitaemia, as well as augmentation of T cell proliferation and IFN-γ production, was evident in PfHGXPRT and sub-patent infected immunised mice. Recipients of CD4+ T cells from PfHGXPRT and sub-patent infection immune mice also showed some degree of protective immunity. PfHGXPRT was shown to induce natural and acquired immunity to P. chabaudi AS. HGXPRT is highly conserved in parasites and humans; therefore, it is essential to define minimal protective epitopes that could be included in a vaccine. Hence, 22 overlapping peptides (termed P1 P22) corresponding to the entire P. chabaudi AS HGPRT sequence were used to define minimal protective epitopes. Following immunisation of mice with seven pools of peptides (P1 P3, P4 P6, P7 P9, P10 P12, P13 P15, P16 P18 and P19 P22), three immunogenic peptides (P11, P13, and P17), which stimulated significant proliferative and IFN-γ responses were chosen for immunisation studies. Peptide P9 (position 76-95 from N-terminal), which induced the highest IFN- levels during P. chabaudi AS infection was also included in the pool of peptides. Mice immunised with P9, P11, P13 and P17 had significantly decreased parasitaemia. Antibody mediated immunity had a partial effect on suppressing parasite growth. CMI, on the contrary, played a central role in adoptively transferred protection by significantly reducing parasitaemia and prolonging survival of recipient SCID mice. Strong T cell proliferation and IFN- secretion were also detected after stimulation of splenocytes from immune mice with P. chabaudi AS antigen. CMI response was significantly increased after immunisation with the peptides followed by sub-patent infection. The findings that four short epitopes of HG(X)PRT confer strong CMI protection suggest that homologues of such epitopes could be included in a multi-component malaria vaccine.

Thesis (Ph.D.)--Murdoch University, 2004. / Thesis submitted to the Division of Health Sciences. Bibliography: leaves 214-249.

Malaria remains an important cause of morbidity and mortality among children in sub-Saharan Africa despite recent reductions in malaria incidence in some parts of Africa. Current control tools face threats such as the emergence of drug resistant parasites and insecticide resistant mosquitoes. A malaria vaccine is needed to complement and/or replace existing tools in order to achieve better malaria control and eventually eliminate the disease. RTS,S/AS01E is the most clinically advanced pre-erythrocytic malaria vaccine candidate and is currently being tested in a phase III trial. The short-term efficacy of RTS,S/AS01E is known but the duration of protection is unknown. Furthermore, although RTS,S is protective, it is unclear which immunological assays predict efficacy: hence there are no known correlates of vaccine-induced protection against clinical malaria. In a randomized controlled trial, I assessed the efficacy of RTS,S/AS01E in children (5-17 months old) residing in Kilifi, Kenya, over 4 years of follow-up and determined the correlates of protection against clinical malaria. In order to examine the effect of variations in malaria exposure on vaccine efficacy, I developed an individual marker of malaria exposure calculated as distance-weighted prevalence of malaria infection within 1 km radius of every child. Over 4 years of follow-up, RTS,S/AS01E had an efficacy of 29.9% (95%CI: 10.3% to 45.3%, p=0.005) and 16.8% (95%CI: -8.6% to 36.3% p=0.18) against first and all malaria episodes, respectively (by intention to treat analysis). Vaccine efficacy waned over time and with increasing malaria exposure. RTS,S/AS01E efficacy was 43.6% (95% CI, 15.5 to 62.3) in the first year but was -0.4% (95% CI, -32.1 to 45.3) in the fourth year. Vaccine efficacy was 45.1% (95%CI 11.3% to 66.0%) among children with lower than average malaria exposure index, but 15.9% (95%CI -11.0 to 36.4%) among children with higher than average malaria exposure index. Despite waning in efficacy, RTS,S/AS01E averted 65 cases of malaria per 100 vaccinated children, with more cases averted among the children in the higher malaria-exposure cohort (78 cases per 100 vaccinated children) than those the low exposure cohort (62 cases per 100 vaccinated children). RTS,S/AS01E induced high titres of anti-CS protein antibodies and CD4+ T cell but not CD8+ T cell responses. Anti-CS antibody titres and the frequency of TNF-α producing CD4+ T cell responses were independently associated with protection from clinical malaria, and the combination of both anti-CS titers and TNF-α producing CD4+ T cell response satisfied the Prentice criteria for surrogate markers of protection. There was no association between avidity of RTS,S-induced anti-CS protein antibodies and protection from clinical malaria. Conclusions: RTS,S/AS01E efficacy against all episodes is 16.8% over the 4 years of follow-up. The vaccine efficacy wanes over time and with increasing malaria exposure. RTS,S/AS01E-induced TNF-α producing CD4 T cell and anti-CS protein antibody responses were independently associated with protection from clinical malaria. Anti-CS avidity did not predict protection from clinical malaria. Long-term follow-ups of malaria vaccine trials are essential in the evaluation of the longevity of vaccine efficacy.

Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

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.