GroEL is a heat shock protein, involved in the folding of denatured proteins under stressed conditions. Flagellin is a structured protein of flagella, and can undergo phase variation so that at any one time, synthesis of flagellin occurs from either of two genes, <i>fli</i>C or <i>fli</i>B. Porins, of which OmpC is an example, allow passage of nutrients and small molecules through the bacterial cell outer membrane. All of these proteins have previously been described as being immunogenic. In the work described here, antibodies specific to each of these antigens were detected in both innately susceptible and resistant mice after infection with an attenuated strain of <i>S. typhimurium</i>, and after subsequent challenge with the virulent organism, suggesting that they may each be involved in protective immunity. The immunogenicity of a vaccine consisting of these four recombinant <i>Salmonella</i> antigens was assessed in the mouse model. GroEL- and flagellin-specific antibodies were detected in innately susceptible mice after immunisation together with the adjuvant DDA. Moreover, analysis of IgG isotypes showed increased titres of both IgG1 and IgG2a, the latter indicating that cell-mediated immunity had been generated. However, challenge of immunised mice with virulent <i>S. typhimurium</i> demonstrated that the protective efficacy of the vaccine was of only low-level. Immunisation of innately susceptible and resistant mice using a tetravalent DNA vaccine expressing all four antigens resulted in increased antibody against GroEL, FliC and FlijB in both mouse strains. As with the subunit vaccine, IgG isotype analysis showed increased titres of both IgG1 and IgG2 indicting that both Type 2 and Type 1 helper T-cell responses had been elicited. However, considerable variation was observed within immunised mouse groups and so the protective efficacy of the vaccine was not determined.

Outbreaks with strains of methicillin resistant Staphylococcus aureus (MRSA) began in a London hospital in 1982 and continue to be associated with significant morbidity and mortality. These particular strains, termed epidemic methicillin resistant S. aureus (EMRSA), are recognised by their phage type and antibiogram. This study examined the ability of isolates representing EMRSA strains recovered from different outbreaks in England and Wales (EMRSA 1-16) to grow in escalating levels of vancomycin. A series of the EMRSA strains were subjected to passage-selection in vancomycin. Six strains became vancomycin resistant, three became vancomycin intermediate, and seven remained susceptible, which confirmed that EMRSA strains were able to develop resistance to vancomycin in vitro. The vancomycin MICs for the vancomycin-resistant derivatives ranged from 24 -32 ug/ml, were associated with decreased lysostaphin susceptibilities and increased cell wall thickness. The MICs of teicoplanin was also increased. Five of the six vancomycin-resistant derivatives became susceptible to methicillin with instability of the S. aureus mecA gene. There were no significant changes to other antimicrobial agents susceptibility, including Linezolid. During this study an attempt was made to elucidate the development of resistance to vancomycin by studying the heterogeneous phenotype of resistance. Population analysis profiles for the vancomycin-resistant derivatives demonstrated that four of them expressed a heterogeneous subpopulation. Bacteriophage typing failed to type vancomycin-resistant clones, while Pulsed-field gel electrophoresis (PFGE) gave good discrimination. In conclusion, we found that, although vancomycin is usually bactericidal against S. aureus, resistance can be common amongst MRSA isolates. Decreased susceptibility to vancomycin appeared to be a selective or inducible process that increased during persistent sublethal exposure to vancomycin. Thus, if this effect occurs in vivo, then not only is vancomycin therapy of serious EMRSA infection compromised, but the propensity to develop resistance may remain undetected by standard laboratory sensitivity testing.

Staph. aureus continues to be a significant pathogen which has developed the ability to acquire resistance to nearly all known antibiotics, in particularly methicillin resistant Staph. aureus (MRSA), has vastly reduced the number of antibiotics effective for treating staphylococcal infections. Recently, clinical isolates, with reduced susceptibility to vancomycin, the only antibiotic effective against multidrug-resistant MRSA, have been reported from a number of centres around the world. This situation could return us to the pre-antibiotic era, when even minor bacterial infections could be fatal. It may also have a significant impact on hospital procedures, especially in cases involving systemic infections, immunosuppression, and surgery. Clearly an understanding of the mechanisms of bacterial resistance to vancomycin, and the search for new anti-staphylococcal drugs or alternative forms of therapy, are necessary steps before epidemics of untreatable staphylococcal infections emerge. This work aimed to investigate the pathogenesis and treatment of Staph. aureus infections. Studies were carried out at phenotypic, and molecular level. Two clinical isolates of EMRSA-15 (clone A and clone C) were serially propagated in media containing increasing concentrations of vancomycin in order to select resistant mutants. EMRSA-15 clone A, yielded the vancomycin resistant clone B. EMRSA-15 clone C, yielded the vancomycin resistant clone 0, which confirms that MRSA was able to develop resistance to vancomycin in vitro. During this research an attempt was made to elucidate the development of resistance to vancomycin in Staph. aureus by studying the heterogeneous phenotype of resistance. The population analysis profile confirmed that EMRSA- 15 clones A and C were susceptible to vancomycin and there was no evidence of vancomycin-resistant subpopulations in these strains, all bacteria ceasing to grow at vancomycin concentrations above 2IJg/ml. The vancomycin resistant clones B and 0 readily demonstrated heterogeneous vancomycin-resistance phenotypes compared with the parent clones A and C.The EMRSA strains susceptible and resistant to vancomycin were extensively investigated for their susceptibility to other antibiotics. It was found that the vancomycin resistant derivatives had significant increases in the minimum inhibitory concentration of vancomycin and teicoplanin, and in addition the MICs for several other antibiotics were altered. In this study two main approaches were taken to investigate mechanisms by which Staph. aureus acquire resistance to vancomycin at molecular level. First RNA arbitrarily primed polymerase chain reation (RAP-PCR), was used to investigate differentially expressed genes in the vancomycin susceptible EMRSA and in the vancomycin resistant EMRSA. One combination of two primers yielded fingerprints with differentially expressed cDNA. This cDNA was expressed in the presence of vancomycin only and it was predicted that this expression was inducible. The second approach involved the sequencing of the the 23S rRNA. A fragment of about 500bp of the 23S rRNA from the vancomycin susceptible EMRSA and from the vancomycin resistant EMRSA was cloned, sequenced, and compared. Point mutations were observed in the EMRSA vancomycin resistant strain: such mutations were absent in the vancomycin susceptible clinical isolate. In this thesis, recombinant antibody derivatives (scFv) obtained from a phage antibody display library which was produced against an immunodominant protein (ABC transporter) from EMRSA was studied. The effect of such molecules (scFv) when used alone or in combination with antibiotics (erythromycin and chloramphenicol) and vinblastine was assessed. ABC transporters seem to constitute generic antigens, conserved between multiple genera, and the neutralisation of these essential transporter proteins may be able to inhibit a wide variety of microbial import and export functions. The data collected in this study showed that the antibody-phages with greatest activity were: phage 16 in combination with erythromycin, phage 12 when combined with chloramphenicol, and phage X when combined with vinblastine. These results indicated that such phages might be used as an alternative form of therapy for multidrug-resistant MRSA.

Human cytomegalovirus (HCMV) is an important opportunistic pathogen capable of causing severe disease in immunocompromised patients and congenitally infected infants. Human cytomegalovirus glycoproteins (gB, gN, gH, gL, gO and gM) are involved in attachment, penetration, viral entry, cell to cell spread and are also capable of inducing neutralising antibody responses. Polymorphisms within glycoprotein genes show focal areas of variability, which allow the identification of clustered genomic variants or genotypes. Little is known about functional differences between variants of glycoproteins H,N,L,0 and M or the occurrence of specific genotype configurations.

In initial experiments, lines of transgenic parasites were generated in which the MHC II-dependent T cell epitope Moth Cytochrome C (MCC) was expressed within a number of different fusion proteins, to provide a model in which the fate of parasite-derived antigens could be followed in infected DC. MHC II-MCC complex formation in CD and MΦ complex-specific T cell line. Although the fusion proteins were clearly demonstrated to be secreted at high levels, no cell surface staining could be detected with D4 and neither infected DC nor infected MΦ could stimulate T cell proliferation. <i>Leishmania-</i>infected DC were however, shown to efficiently process and present exogenous antigen to T cells <i>in vitro.</i> Alternative strategies were therefore developed to probe the DC:<i>Leishmania</i> interaction in more detail. Investigation into the effect of uptake of EGFP-expressing <i>L. mexicana </i>parasites by different DC cultures <i>in vitro</i> demonstrated that, in the absence of exogenous factors, uptake of <i>L. mexicana </i>amastigotes did not cause activation of DC. Uptake of <i>L. mexicana </i>promastigotes resulted in activation of a small proportion of DC indicating that promastigotes do encode an activation signal, but that this is not sufficient to activate the entire DC population. However, neither <i>L. major </i>promastigotes nor <i>L. mexicana </i>promastigote mutants lacking surface lipophosphoglycan (LPG) activated DC <i>in vitro. </i>Therefore these data suggest that <i>L. mexicana </i>promastigotes encode an activation signal, but that this is not sufficient to stimulate all DC. As the promastigotes which did not activate DC either lacked, or expressed a modified version of, LPG, we propose that LPG is a <i>L. mexicana </i>pathogen-associated molecular pattern (PAMP). The work presented in this thesis demonstrates that infected DC are capable of initiating that anti-<i>Leishmania </i>response <i>in vivo, </i>as they efficiently present antigen to T cells. However, infection <i>per se </i>is not sufficient to activate all DC. These data therefore suggest that during the initiation of an anti-<i>Leishmania </i>T cell response DC are likely to be activated by factors produced in response to injection of parasites by the insect vector, such as pro-inflammatory cytokines.

This thesis presents two novel immunisation strategies against <i>Salmonella enterica</i> serovar Typhimurium (<i>S. typhimurium</i>) in the mouse model. Firstly, mice immunised with multiple antigens in the form of a DNA vaccine have been shown to develop specific humoral and cellular responses to proteins encoded within the vaccine. Secondly, it has been shown that immunisation with multiple cytosolic antigens (CA) of <i>S. typhimurium </i>SL1344, formulated with the adjuvant dimethyl dioctadecyl ammoniumbromide (DDA), induces strong humoral and cellular responses. These responses have been characterised, and their ability to confer protection on mice challenged with a lethal dose of <i>S. typhimurium </i>SL1344 has been investigated. The aim of DNA vaccination is to induce immune responses to protein antigens expressed <i>in vivo</i> by injection plasmid DNA encoding the antigen sequence. Expression library immunisation (ELI) is a new technique that draws on DNA immunisation and can be developed to identify the key antigens of a pathogen that confer protection. ELI has previously been used in a number of disease models in mice and has been used in this thesis as a means of inducing immune responses against an unspecified subset of bacterial antigens. An expression library (EL) of <i>S. typhimurium </i>SL1344 was constructed using a mammalian expression vector encoding EGFP. The expression of <i>S. typhimurium </i>SL1344 antigens forming fusion proteins with EGFP was visualised as green fluorescence in mouse fibroblast cells <i>in vitro. </i>The EL consisted of 140,000 clones of which 14,000 were used for immunisation. The library was administered to both BALB/c and CBA mice to examine the effect of mouse strain and was administered both intradermally (ID) and intramuscularly (IM) to examine the effect of immunisation route. Analysis by Western blot and ELISA showed that humoral responses were induced in both BALB/c and CBA mice. ID and IM inoculation produced similar results in BALB/c mice. Responses included a significant Thl component as judged by the presence of IgG2a in the serum and the secretion of IFN-γ when T cells from peripheral lymph nodes were stimulated by CA in culture.

Rosetting – the binding of parasitized red blood cells to uninfected red cells to form “rosettes” has been associated with severe disease in many studies in Africa. However, a study in Papua New Guinea found no association between resetting and severe disease. It remains unclear whether resetting is a cause or correlate of parasite virulence. Rosetting is mediated by the interaction of at least one parasite protein: <i>Plasmodium falciparum</i> erythrocyte membrane protein one (PfEMP1) on the infected erythrocyte surface and a variety of host red cell surface receptors including complement receptor one (CR1). We reasoned that if resetting were a cause of severe disease we would be able to identify protective polymorphisms in the CR1 gene. Here we show that CR1 deficiency is extremely common among individuals from malaria endemic populations in Papua New Guinea. This deficiency is associated with previously reported polymorphisms in the CR1 gene and unexpectedly with alpha-thalassaemia, a red cell disorder that occurs in up to 90% of Melanesians. We tested the hypothesis that CR1 polymorphisms protect against severe malaria by genotyping samples from a case control study for polymorphisms in the CR1. We found that both CR1 deficiency alleles and alpha-thalassaemia protect against severe malaria. We wished to test the hypothesis that resetting was not associated with severe disease in Papua New Guinea because of widespread CR1 deficiency preventing the formation of rosettes strong enough to withstand sheer forces in the circulation. Studies on field isolates from Papua New Guinea showed that resetting was unusual in this population, rarely mediated by binding to CR1 and not associated with disease severity. This suggests that CR1 is essential for physiologically significant resetting associated with severe malaria. We have therefore identified a new malaria resistance gene and provided compelling evidence that CR1 mediated resetting is an important virulence phenotype and a potential target for drug vaccine development.

The research outlined in this thesis is mainly designed to develop a technology of producing a DNA prime-protein boost vaccine against HIV-1 infection. To reach this aim 1.7kb fragments encoding gp120 antigens deriving from two groups of British HIV-1 infected persons (one consisting of homosexual individuals from Edinburgh, Newcastle and Belfast and the other consisting of haemophiliac patients from Edinburgh who became infected from a common batch of Factor VIII) were PCR amplified and subsequently subcloned into a cloning vector (pGEM T). A mammalian expression vector (pSRHS) was modified in order to include a polylinker to allow the transfer of the 1.7-kb fragments from pGEM T to pSRHS. The recombinant clones were identified and the gp120 genes were expressed in mammalian cells (COS cells) by lipofectin protocol. The functional clones (i.e. those that contained intact open reading frames), were selected and their associated gp120 antigens were quantified by an 'in house' ELISA method. Equivalent amounts of the gp120 antigens were used in an anti-gp120 ELISA to estimate the extent of recognition by the IgG antibodies from autologous and heterologous sera. The nucleic sequences of the functional clones were obtained and some properties such as their predicted NSI/SI phenotype, co-receptor usage and glycosylation sites were analysed. The phylogenetic relationship between the sequences derived from both cohorts was computed and the extent of gp120 antigen recognition by the IgG antibodies from the autologous and heterologous sera was analysed in conjunction with their degree of relatedness. As a conclusion of this study, a high degree of cross-reactivity was noted between antigens and sera, the extent of the recognition of the antigens by the sera was given by the patients' immune status. No significant difference in recognition of the gp120 antigens by sera was observed. This result points towards the potential usage of a cocktail of such DNAs and their corresponding gp120 antigens as a DNA prime-protein boost vaccine.

Preliminary experiments employing the Mu<I>dlux</I> reporter system indicated that the <I>ahp</I> locus was osmotically sensitive in <I>S. typhimurium. </I>Studies on other bacteria supported this view. The affect of the osmotic environment of the cell upon the expression of <I>ahp</I> was therefore addressed in greater depth. The subsequent use of immunoblotting techniques conclusively demonstrated that chromosomal expression of the <I>ahp</I> locus was not affected by the osmotic environment surrounding the cell. Instead, the Mu<I>dlux</I> element was found to alter the natural behaviour of the <I>ahp</I> promoter in such a way that it adopted an osmotically-regulated status, and this mode of regulation appeared to override regulation via the normal hydrogen peroxide-inducible mechanism. <I>S. enterica </I>is an intracellular pathogen which is capable of surviving within macrophage cells. Macrophages are equipped with an arsenal of anti-microbial effector mechanisms, including a respiratory burst which generates reactive oxygen metabolites. Since <I>ahp</I> had previously been shown to respond to the respiratory burst of macrophages, this study also assessed the role of oxidative stress resistance genes in the virulence of <I>S. typhimurium. </I>Strains of the mouse pathogen Sl1344 were constructed in which the <I>ahp</I> and <I>oxyR</I> loci were disrupted and their virulence was assessed in LD<SUB>50</SUB> studies. Disruption of the <I>ahp </I>or <I>oxyR</I> loci was found to have no effect upon the gross virulence of SL1344 for mice, suggesting that these loci were not essential for survival within the macrophage. The ability to develop immunity against infection by <I>S. typhimurium </I>is thought to correlate with the development of immunity against bacterial antigens which are expressed <I>in vivo. </I>As a further part to this study, the immunological responses of mice to two <I>S. typhimurium-</I>derived polypeptides, AhpC and GroEL, following cloning and overexpression of these proteins, were examined. Mice previously infected with an attenuated strain of <I>S. typhimurium </I>were shown to elicit significant delayed-type hypersensitivity reactions following subcutaneous injection of these polypeptides 33 and 104 days post-infection. Moreover, AhpC-and GroEL-specific antibodies were detected during the course of infection of mice with <I>S. typhimurium. </I>

In this thesis, three aspects of parasite life history were investigated: the trade-off between asexual growth and reproduction, the virulence of infections and the sex ratio of transmission stages. All three, and the frequency of mixed infections in a population itself, are fundamentally linked to the transmission and pathology of malaria, the reduction of which is the aim of all current control programmes. An experimental system of the rodent malaria <I>Plasmodium chabaudi </I>in mice allows controlled mixed-infections of distinct clones to be initiated and was used to test theoretical predictions. Genetic and immunological techniques were used to monitor the parasite population throughout the life cycle. One of the main findings from the experimental system was also tested in field populations of the human malaria <I>P. falciparum. </I>Compared to single-clone infections, mixed-clone infections of <I>P. chabaudi </I>showed increased transmission rates, higher virulence and in some experiments less female-biased gametocyte sex ratios. These results are in line with theoretical expectations, but in several cases the underlying mechanism responsible was not entirely consistent with that assumed by theory. The relative transmission rates of two clones in a mixed infection were not predicted by their relative starting inocula or their relative asexual parasite abundances four days before transmission was measured. The minority clone in a mixed-clone infection inoculum transmitted at least as well, and often far better, than it did as a single-clone control. Asexual parasite population sizes were often poor predictors of transmission and virulence. However, the data show common patterns across experiments which help to elucidate patterns of interactions between parasite genotypes and the host. The results are consistent with a strong strain-specific component to host immunity, with a suggestion that immune responses to mixed-genotype infections may be more costly for the host than those directed against single-genotype infections. The relevance of all experimental results to the control of human malaria parasites is discussed.