The aim of this project was to employ phage-display libraries to investigate epitopes recognised by antibodies directed against the hepatitis B virus surface antigen (HBsAg). The experiments were based upon a library comprising a pool of filamentous phage (fusion-phage) which displayed random hexapeptides on the surface of the particles. Antibodies of monoclonal and polyclonal origin were used for selection of phage that bound the antibodies via their exposed peptides. In the case of the monoclonal antibody, the phage ligands selected showed highly related amino acid sequences with a few distinctively conserved residues. These residues could be aligned with the primary sequence of the PreS1 domain of HBsAg thus precisely locating the epitope recognised by this monoclonal antibody. A quantitative assay, adapted specifically for use with fusion-phage particles, revealed binding constants in the nM range for the interaction of the fusion-phage with the antibody. Comparison of the binding constants of phage with divergent amino acid sequences and competition binding studies with synthetic peptides defined the epitope recognised by this antibody with high-resolution. A similar approach was applied to polyclonal serum IgG from a chimpanzee that had been vaccinated with HBsAg. It has been demonstrated that the phage-display approach can provide high-resolution epitope mapping for monoclonal antibodies. Quantitative values for the interaction in solution between fusion-phage representing this epitope and the antibodies have been obtained readily by means of a simple assay based upon phage titre. Further, even within the complex context of a polyclonal serum individual epitopes or at least elements of them could be identified, thus indicating immunologically important areas of this antigen.

No description available.

The design and synthesis of discontinuous epitopes of HIV-1 gp120 has been investigated. This has been exemplified by the design of a peptide to mimic a discontinuous but conserved region of gp120 containing residues important for CD4 binding. This peptide was synthesised using the Fmoc strategy of solid phase peptide synthesis. The problem of aspartimide formation as a side product in the synthesis was observed and this process was addressed in more detail. Polyclonal antisera were raised in mice against the purified peptide and the antipeptide antibodies characterised. Initial evidence suggests this peptide binds to CD4 and induces apoptosis.

The complement protein (CP) molecule (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CP modules comprise specific interaction sites for other molecules. Hence the orientation in space of a CP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. The Vaccinia virus complement control protein (VCP) is a complement regulator composed in its entirety of four tandemly arranged CP modules. The central two modules of this protein have been successfully expressed in <i>Pichia pastoris </i>and the structure of the modules, numbers 2 and 3 (VCP~2, 3), solved using NMR spectroscopy. Each module has a typical CP module structure and the two modules do not share an extensive interface. Inspection of fifty structures calculated independently on the basis of the NMR-derived data revealed that the inter-modular orientation does not vary much implying that the 40 linker-to-module NOEs limit the possible movement of the two modules relative to one another during the structure calculation. Module 2 contains a five residue insertion and has a more elongated appearance than module 3. Module 3 appears bulkier due to the laterally protruding hypervariable loop. Module 3 from VCP~2,3 differs a little in structure from module 3 from the VCP~3, 4 module pair. The structure of VCP~2, allowed reconstruction of VCP~2,3,4 and provides a means of gaining structural information about VCP as a whole using a dissect and rebuild strategy. A recent crystal structure of intact VCP exhibits a somewhat different orientation of the two central modules.

The versatility of bacteriophage lambda as a vector for the amplification of DNA fragments has been limited by the technical difficulties involved in preparing vectors possessing one or two targets for particular restriction enzymes. In this thesis, the use of 'adapter fragments', i.e., short, naturally occurring DNA fragments terminated by the recognition sequence for a different restriction enzyme at each end, has been studied. Such fragments have been employed to insert a DNA fragment produced by R.T digestion into a vector R.Hin III target. The need to isolate adapter fragments has been obviated by using genetic or physical screening or selection for the insertion of adapter fragments. Two DNA fragments of interest from plasmid recombinants have been transferred to lambda recombinants using these techniques.

The synthesis of IH1, a peptide designed to mimic a discontinuous epitope on HIV-I gp120, is reported. The aspartimide rearrangement inherent in this sequence, and in the peptide GC1, has been studied and reduced to low levels. The syntheses of variants of peptide GC1, containing differing number of residues found to be important for CD4 binding, have also been achieved. Thus peptides containing one, two, three and four residues necessary for high affinity binding have been synthesised. In addition a peptide has been synthesised which incorporates a synthetic β turn moiety other than the Cys-Val-Cys bridge present in GC1. Polyclonal sera raised to these peptides and their CD4 binding properties have been investigated. IH1, the peptide containing four residues responsible for high affinity binding to CD4, has also been shown to interact with receptors on the surface of CD4<SUP>-</SUP> cells. This non-CD4 recognition has been investigated utilising a photolabelled chemokine, MIP-1α. Results indicate that this binding involves interaction with CC-CKR5, a MIP-1α binding site thought to be involved in HIV-cell fusion.

No description available.

Lentiviral vectors (LVs) are successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins during replication of lentivirus HIV-1 helped to understand virus assembly and budding. Knowing what cellular proteins interact with viral proteins and identifying interactions that promote formation of functional particles can be valuable for improving LV production in Gene Therapy. The cellular protein composition of LVs produced by two different methods was compared, the transient transfection system producing vectors pseudotyped with the VSV-G envelope and a stable producer cell system producing vectors pseudotyped with the non-toxic retroviral envelope, RDpro. Lentiviral vectors were purified using size exclusion chromatography (SEC). The number of purified LVs produced by transient transfection was six fold higher compared to stably produced particles. For linear ion trap-orbitrap tandem mass spectrometry (LTQ-MS/MS) a comparable amount of SEC purified LVs was analysed, detecting a smaller number of cellular protein species in stably compared to transiently produced vector samples. The greater numbers of host proteins in purified transiently produced samples may due to the presence of co-purified VSV-G vesicles. On the other hand, a large number of proteins we identified had also been detected in studies of wild type viruses and HIV-1 derived vectors indicating a role in vector formation, such as viral protein transport to the assembly site in producer cells. The potential role in LV particle production of selected identified proteins was assessed. Whilst some proteins that have been detected in studies on wild type HIV-1 were found in all our samples, such as ALIX, AHNAK was unique to stably produced, RDpro pseudotyped vector samples, and thus selected for further investigation. In summary, knock down of ALIX, AHNAK and TSG101 host cell proteins in vector producer cells did not result in a significant difference in vector production.

Inhibition of the Human Immunodeficiency virus type-1 (HIV-1) protease enzyme blocks HIV-1 replication. Protease inhibitor drugs have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow the progression to Acquired Immune Deficiency Syndrome (AIDS). However, mutations readily and rapidly accrue in the protease gene resulting in a reduced sensitivity of the protein to the inhibitor. In this thesis, molecular dynamics simulations (MDS) were run on HIV proteases complexed with the protease inhibitor saquinavir, and the strength of affinity calculated through MMPBSA and normal mode analysis. We show in this thesis that at least 13 residues can be computationally mutated in the proteases sequence without adversely affecting its structure or dynamics, and can still replicate the change in binding affinity to saquinavir caused by said mutations. Using 6 protease genotypes with an ordered decrease in saquinavir sensitivity we use MDS to calculate drug binding affinity. Our results show that single 10ns simulations of the systems resulted in good concurrence for the wild-type (WT) system, but an overall strong anti-correlation to biochemically derived results. Extension of the WT and multi-drug resistant (MDR) systems to 50ns yielded no improvement in the correlation to experimental. However, expansion of these systems to a 10-repetition ensemble MDS considerably improved the MDR binding affinity compared to the biochemical result. Principle components analysis on the simulations revealed that a much greater configurational sampling was achieved through ensemble MD than simulation extension. These data suggest a possible mechanism for saquinavir resistance in the MDR system, where a transitioning to a lower binding-affinity configuration than WT occurs. Furthermore, we show that ensembles of 1ns in length sample a significant proportion of the configurations adopted over 10ns, and generate sufficiently similar binding affinities.

The research outlined in this thesis was primarily designed to study the quantitative and qualitative variability of plasma viral RNA during the course of HIV-1 infection. The quantitative aspect involved the development of a highly sensitive and reliable RNA-based PCR method which has been used to detect and quantify HIV RNA load directly from patient materials (plasma, serum). High levels of plasma varaemia were observed during the first stage of HIV-1 infection and considerably higher than those observed in symptomatic patients. However, the high viral loads during this period are transient, and a marked drop in virus quantity was observed with the development of anti-HIV specific immune response. On average, HIV RNA was more abundant in the plasma of patients with more advanced disease compared to asymptomatics. However, the observation of persistent high levels of HIV RNA in some asymptomatic patients suggests that viral replication continues throughout the course of HIV infection and that there is no 'latent' period to correspond with that observed with clinical progression. Extensive studies of sequential variation in the HIV-1 envelope gene constitute the qualitative element of this research and have revealed that there are complex evolutionary patterns. No sequence variation was observed in the V3 and V4 regions in any of the samples collected prior to or immediately after seroconversion, although variation was present in the <i>gag</i> gene at this time. Such an observation led to the suggestion that there is a strong selection for the most rapidly replicating viral variants before the immune response is mounted. However, along with the development of specific anti-HIV immune response, the pattern and process of HIV-1 sequence variation changes. Rapid changes in the viral population were observed within weeks of seroconversion. Phylogenetic analysis of the V3 sequences from patient 82 identified several evolutionary lineages of virus variants after 3 years of infection, only two of which persisted and subsequently reached high frequency.