Magister Public Health - MPH / A qualitative descriptive study approach was used to gather the required information. The sample for this study was drawn from an existing group of volunteers who participated in the vaccine trial at Mbeya Medical Research Centre in 2006-2007. A purposive sampling method was used to select respondents because they had had experience of being participants in a HIV vaccine trial. Twenty audio recorded in-depth interviews were conducted. The interviews were conducted at the clinic during their routine follow up visits. An open ended interview guideline was used to guide the discussion to elicit the required information from the respondents. The data was transcribed, translated and then analyzed by both content and thematic approach. Ethical procedures were observed, including getting permission from the local ethical committee in Mbeya region and participants were given an informed consent form to read and sign before starting the interview. / South Africa

HIV-1 is the causative agent for AIDS and has been a major focus of research for the past two decades. Though there is a combination therapy in place known as the “Highly Active Anti-Retroviral Therapy” (HAART), its usefulness is confounded by the generation of escape mutants, a host of side effects, and its prohibitive cost. The most useful alternative would be the prevention of infection by vaccination. Vaccine research has been focused on the use of recombinant protein sub-units of the virus or combinations thereof to elicit a neutralizing response against the virus. These approaches have mostly resulted in a failure to generate broadly cross reactive neutralizing response against primary strains of the virus. The work reported herein is aimed at designing a rigidified version of gp120/gp120 derivatives and understanding the scope of the various antigenic regions in gp120 in generating a neutralization response. Chapter one discusses some general features of the virus and the immune system. The general nature of AIDS, its spread and its immunological characteristics are also described in this chapter. Chapter two discusses the design and NMR structural analysis of gp120 bridging sheet peptide mimics in methanol and water. The structure of gp120 can be loosely divided into two domains (the outer domain and the inner domain) that are linked together by a discontinuous four stranded antiparallel beta sheet known as the bridging sheet. The bridging sheet is known to overlap with the coreceptor binding site of gp120 and hence is a suitable target for designing virus-entry inhibitors. 17b, a neutralizing antibody isolated from an infected individual, is known to bind to this region of gp120. Our aim in this part of the work was to design a four stranded antiparallel beta sheet, based on the sequence of the bridging sheet, that would contain most of the residues involved in 17b binding. NMR and CD studies confirmed that the peptide was well structured in methanol but the structure was largely lost on addition of aqueous solvent. A small population of the peptide was found to be well-folded in aqueous solution. Chapter three discusses the design and characterization of a gp120-CD4D12 single chain. It is well known that the conformation of gp120 changes upon binding CD4 to expose cryptic epitopes, known as CD4i epitopes. In this work we report the generation of a single chain gp120-CD4 construct that has the cryptic epitopes exposed. The construct bound to 17b, a conformation specific antibody against the bridging sheet of gp120, a cryptic epitope, as well as a non-covalent complex of gp120:CD4D12. There was also very insignificant secondary structural change in gp120 upon complex formation with CD4D12 as observed by CD spectroscopy. Immunological studies with DNA and protein vaccination in guinea-pigs indicated that though 17b like antibodies are generated after immunization, they did not contribute towards the neutralization of primary isolates of the virus. It was also observed that it was the anti-CD4D12 antibodies that were responsible for the neutralization by the sera. These studies indicated towards the inability of the bridging sheet to generate effective neutralization response in case of vaccination with gp120/CD4 complexes. Chapter four discusses the design of a mimic of the gp120/CD4 complex. Since it was seen from our previous work that gp120/CD4 complexes generate a large fraction of antiCD4 antibodies and hence are unsuitable for vaccination purposes, we generated a construct with the minimal binding region of CD4. The small fragment of CD4 spanning from 21st residue to 64th residue was inserted in the V1/V2 loop of gp120. The insertion site was designed based on the region of gp120 closest to this fragment and capable of tolerating insertions. This protein did not bind to 17b as well as gp120/CD4 complex but showed a higher binding compared to full length gp120. Further immunological characterization with this protein revealed that it was not capable of generating neutralizing antibodies against the virus. Chapter five discusses the design and execution of a SPR based solution phase competition experiment to find the solution phase binding constant of CD4 and CD4 analogs to gp120. A major problem during the analysis of binding data obtained by SPR is the accurate determination of Rmax, a parameter needed to obtain an accurate equilibrium dissociation constant. In this chapter we have developed a binary as well as a ternary solution phase SPR based assay to accurately determine a solution phase equilibrium binding constant. The binding constants were determined for gp120 binding to CD4D12 and other CD4 analogs. To confirm the validity of the assay, a control antigen:antibody interaction whose equilibrium dissociation constant has been determined by other methods has been used as a test case. Chapter six discusses the design and characterization of V3 peptides inserted in the loop regions of E. coli Thioredoxin (Trx). Trx has earlier been used to display random peptide libraries between the 33rd and the 34th residue. We have constructed three constructs where the peptide has been inserted between the 33rd and 34th residue, between the 74th and 75th residue and between the 84th and 85th residue. The insertion between 74th and 75th position (74V3Trx) was found to be superior to the other two and would be a suitable alternative for display of a random peptide library. The binding of these constructs to 447-52D, a V3 peptide specific antibody was characterized. These were also characterized immunologically, and 74V3Trx was found to generate weakly neutralizing activity against the MN strain of HIV-1. Competition experiments with 447-52D with these sera indicated that there were antibodies generated that could compete out 447-52D binding to gp120 but not in sufficient concentration to provide broad neutralization. Appendix 1 discusses the rational design of disulfides to stabilize proteins based on the analysis of naturally occurring disulfides. In our attempts to design a rigidified version of gp120 we had designed disulfides in gp120 based on its crystal structure. Many of these were disulfides that would span antiparallel adjacent strands. In order to improve the design principles, we analyzed naturally occurring disulfides that span antiparallel adjacent strands and characterized them in terms of their positional preference in a beta sheet. It was found that these disulfides mostly occur on edge strands and are found exclusively between non-hydrogen bonded registered pairs of adjacent antiparallel strands. Mutagenesis on Thioredoxin was performed to verify our results. It was found that disulfides designed between the non-hydrogen bonded pairs of antiparallel strands could significantly stabilize the protein whereas the ones between hydrogen bonded pairs destabilized the protein.

Des données complexes sont fréquentes dans les essais cliniques récents et nécessitent des méthodes statistiques adaptées. La recherche vaccinale du VIH est un exemple d’un domaine avec des données complexes et une absence de critères de jugement validés dans les essais précoces. Cette thèse d’Université concerne des recherches méthodologiques sur la conception et les aspects statistiques des essais cliniques vaccinaux du VIH, en particulier sur les critères de jugement d’immunogénicité et les schémas d’essai de phase I-II. A l’aide des données cytokiniques multiplex, nous illustrons les aspects méthodologiques spécifiques à une technique de mesure. Nous proposons ensuite des définitions de critères de jugement et des méthodes statistiques adéquates pour l'analyse des données d'immunogénicité multidimensionnelles. En particulier, nous montrons l’intérêt des scores multivariés non-paramétriques, permettant de résumer l’information à travers différents marqueurs d’immunogénicité et de faire des comparaisons inter- et intra-groupe. Dans l’objectif de contribuer à la conception méthodologique des nouveaux essais vaccinaux, nous présentons la construction d’un schéma d’essai optimisé pour le développement clinique précoce. En imbriquant les phases I et II d’évaluation clinique, ce schéma permet d’accélerer le développement de plusieurs stratégies vaccinales en parallèle. L’intégration d’une règle d’arrêt est proposée dans des perspectives fréquentistes et Bayesiennes. Les méthodes mises en avant dans cette thèse sont transposables à d’autres domaines d’application avec des données complexes, telle que les données d’imagerie ou les essais d’autres immunothérapies. / Complex data are frequently recored in recent clinical trials and require the use of appropriate statistical methods. HIV vaccine research is an example of a domaine with complex data and a lack of validated endpoints for early-stage clinical trials. This thesis concerns methodological research with regards to the design and analysis aspects of HIV vaccine trials, in particular the definition of immunogenicity endpoints and phase I-II trial designs. Using cytokine multiplex data, we illustrate the methodological aspects specific to a given assay technique. We then propose endpoint definitions and statistical methods appropriate for the analysis of multidimensional immunogenicity data. We show in particular the value of non-parametric multivariate scores, which allow for summarizing information across different immunogenicity markers and for making statistical comparisons between and within groups. In the aim of contributing to the design of new vaccine trials, we present the construction of an optimized early-stage HIV vaccine design. Combining phase I and II assessments, the proposed design allows for accelerating the clinical development of several vaccine strategies in parallel. The integration of a stopping rule is proposed from both a frequentist and a Bayesian perspective. The methods advocated in this thesis are transposable to other research domains with complex data, such as imaging data or trials of other immune therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.