None of the nematodes that cause human falarial disease readily establish and develop in laboratory mice. However the rodent filarial nematode <i>Litomosoides sigmodontis</i> can complete development to patency in BALB/c inbred mice. The studies in this thesis have utilised this rodent model to study factors that influence the establishment of filarial infection. Filarial nematodes evoke strong type 2 host immense responses characterised by the cytokine interleukin 4 (IL4). By infecting mice that are unable to produce IL4 we have discovered that IL4 can mediate resistance to the establishment of <i>L. sigmodontis</i> infection. Infections of chimeric mice that can only produce IL4 from the innate or the acquired immune response indicate that IL4 needs to be produced from both arms of the immune system to confer protection. We have also studied the impact of co-infecting protozoan pathogens that induce strong type 1 responses. Co-infection with the protozoan parasites <i>Plasmodium chabaudi</i> or <i>Leishmania major </i>resulted in the accelerated death of established adult <i>L. sigmodontis</i>. Immunological analysis indicates that this was correlated with a decrease in type 2- like responses against <i>L. sigmodontis</i> parasites in both cases. However we did not detect any increase in type 1-like responses against <i>L. sigmodontis</i> in either of these studies. Finally we investigated the role of the filarial intracellular bacteria <i>Wolbachia. </i>We established that anti-<i>Wolbachia</i> immune responses do occur in human filarial infection, in individuals not treated with anti-filarial drugs. Using general linear modelling we determined that anti-<i>Wolbachia</i> surface protein (WSP) antibodies are generated mainly by the L3 stage. In support of this, analysis of life cycle stages of <i>L. sigmodontis</i> indicate that, per gram of nematode material, the L3 stage evokes by far the greatest antibody responses against WSP. Additionally we have shown that anti-WSP immune responses can, in some circumstances, promote the establishment of primary <i>L. sigmodontis</i> infection.

The principal target of antibodies that can neutralise HIV-1 infection <I>in vitro</I> is the surface envelope protein, encoded by the envelope gene (<I>env</I>). The <I>env</I> gene evolves rapidly during the course of infection, producing a multitude of variants, many of which show different sensitivities to neutralisation by antibodies. The work described in this thesis, examines the hypothesis that the selection of antibody-resistant variants of the <I>env </I>gene is a strong force shaping the evolution of the <I>env</I> gene, a process that allows the virus to persist in the face of a strong immune response. In order to examine the antibody-response to the <I>env</I> gene, 19 HIV-1 clones were constructed by replacing the <I>env</I> gene of the infectious molecular clone pHXB2-D, with homologous <I>env</I> sequences obtained from a single individual. The 19 <I>env</I> genes were obtained directly from patient peripheral blood mononuclear cell DNA using the polymerase chain reaction. The sequences of the <I>env</I> genes represent the predominant types found in the plasma at seroconversion and in five subsequent years of infection within this individual. No inactivating mutations were found following the partial sequencing of each <I>env</I> clone. Following the introduction of the recombinant proviral clones into human T-cell cultures, only five of the 19 showed evidence of the production of high-replicating viruses. Viruses from these clones induced cell-to-cell fusion (or syncytia), and were capable of growth in a variety of human T-cell lines. With the remaining 14 clones, three showed evidence of low level replication in human peripheral blood mononuclear cells, but failed to replicate in T-cell lines and did not induce the formation of syncytia. The remaining 11 clones but did not show evidence of productive infection. In conclusion, despite being taken directly from an infected patient, only a small proportion of <I>env</I> genes were found to support productive infection <I>in vitro. </I>

This thesis seeks to address the mechanisms that underlie the development of the profound cellular hyporesponsiveness in filariasis, and the relationship of this to the development of Th2-type responses. These questions were addressed using a murine model of infection with <I>Brugia malayi</I>, one of several species of parasitic nematode that are responsible for filarial disease. In this model, mice develop strong Th2 responses. The findings in this thesis relate to both parasite and host factors involved in the development of immune suppression. Adult and infective larval parasites, but not microfilaria, generated down-regulatory host cells. The excretory/secretory products of the adult parasite were sufficient to induce the generation of PEC that block proliferation. Suppressor cell generation correlated directly with systemic IL-4 production by the host. We were able to demonstrate that there was a direct link between host production of IL-4 and the generation of suppressive PEC, as IL-4-deficient mice failed to induce proliferative block. However, implanted IL-10-deficient mice resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Experiments using transwell membranes or formaldehyde-fixing of PEC indicated that suppression was not effected by a soluble mediator, and required cell-cell contact. These data suggest that the mechanism of suppression exhibited by down-regulatory cells from filarial-infected mice appear to be unlike those previously described in other infectious disease systems, being dependent on host IL-4 production, and effected through a cell-contact-mediated mechanism.

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No description available.

No description available.

Bunyamwera orthobunyavirus (BUNV) is the prototype for the family Bunyaviridae. BUNV has a tripartite RNA genome of negative polarity composed of the large(L),medium (M)and small(S)segments. Each segment contains an open reading frame (ORF) flanked by untranslated regions (UTRs). The eleven terminal nucleotides are conserved between the three segments while the internal regions are unique. The UTRs play an important role in the virus life cycle by promoting transcription, replication and encapsidation of the viral genome. The work presented in this thesis explores UTRs plasticity and examines ways to engineere attenuated viruses by modifying only their UTRs. Using reverse genetics, mainly two ways of attenuation were explored: rescue of viruses either carrying deletions within their 3' and/or 5' UTRs in all three segments, or of viruses carrying one segment bearing heterologous UTRs. Both approaches resulted in virus attenuation in tissue culture, with viruses producing smaller plaques or even no plaques, and growing to lower titres than wild-type BUNV. Through serial passage, viruses were shown to regain some level of fitness while the mutations introduced in the UTRs proved to be stable. Thus, to investigate the mechanism behind fitness recovery, the nucleotide sequence of the entire genome of viruses with deletions in their UTRs was determined. Amino acid changes were observed in the viral polymerase (L protein) of most mutant viruses and the vast majority of the amino acid changes occured in the C-terminal region. The function of this domain is unclear to date, however data obtained using a mini-replicon assay suggest that the C-terminal domain of the L protein might be involved in UTR recognition. Full genome sequencing also allowed the identification of an amino acid mutation within the polymerase that resulted in a temperature sensitive phenotype when introduced in an otherwise wild-type BUNV. Thus, it was shown that mutations introduced within the UTR regions of the genome were stable through serial passage and resulted in attenuation. Such a strategy could be used in combination with mutations of the ORF to design live-attenuated vaccines against serious pathogens within the family Bunyaviridae.

Expression of the amidase operon of Pseudomonas aeruginosa is regulated by the AmiC and AmiR proteins using a version of the bacterial two-component signal transduction system. The negative regulator, AmiC is the amide ligand sensor and regulates activity of AmiR by a steric hindrance mechanism. AmiR, the response regulator functions as a transcription antitermination factor. Once released from the AmiC/AmiR complex, AmiR binds to the operon leader transcript to prevent formation of the rho-independent terminator thus allowing transcription of the entire operon. AmiR has a CheY-like N-terminal response regulator receiver domain without the conserved phosphate-acceptor residues and a long coiled-coil C-terminal domain terminating in a three-helix bundle ANTAR domain. Within this family are a number of highly conserved residues. These residues have been changed by site-directed mutagenesis and the mutants tested for antitermination activity by amidase assay. Random mutagenesis has also revealed key residues in this domain, changes which led to reduced antitermination efficacy. To define the minimal transcript length sufficient for AmiR antitermination, constructs have been made and analysed containing leader region mutations and deletions. The results show that the encoded leader ORF has no biological function and that sequences upstream of the previously defined L recognition region are not necessary for AmiR-dependent antitermination. The ANTAR domain has been expressed and purified as a cleavable, GST tagged fusion protein in E. coli. Dynamic light scattering data and gel filtration elution profiles of ANTAR suggests this domain is a dimer in solution. The ANTAR-RNA interaction was investigated in vitro using band shift assays. Radiolabeled in vitro transcribed ami leader RNA was incubated with the ANTAR protein under various buffer conditions. The mixtures were analysed by PAGE to monitor the formation of protein-RNA complexes. Amidase assays were also performed with the ANTAR domain for analysis of antitermination activity in vivo. These results show that the ANTAR domain alone is not sufficient for antitermination in vivo or binding to the RNA in vitro.

No description available.

No description available.