The HIV-1 envelope glycoprotein (Env) is the primary focus of prophylactic HIV vaccine development. However, the unusually low density of Env spikes on the virion (≈14 spikes/virion) is unfavourable for eliciting high titre, long-lasting antibody responses. It is possible that increasing the Env spike density of particulate vaccine candidates generated by protein body formation or via the display of Env on nanoparticles could improve the induction of long-lasting neutralising antibodies (NAbs). For this thesis, two different nanoparticle approaches were therefore investigated. The HIV-1 Env sequence used for both approaches was derived from the superinfecting subtype C CAP256 virus. This was truncated to remove the transmembrane domain, and engineered to contain a flexible linker (FL) in place of the furin cleavage site and an I559P mutation to generate soluble, stable and cleavageindependent gp140 proteins. The first approach investigated the impact of genetically fusing a 27 kDa proline-cysteine-rich domain of the ɣ-zein maize seed storage protein - Zera® - to either the N- or C-terminus of CAP256 gp140. Fusion of Zera® to a protein of interest can promote the self-assembly of large protein bodies (PBs) containing the protein of interest, thereby improving yields of the recombinant protein and enabling easy isolation using gradient ultracentrifugation. The purification of Zera-induced Env PBs from infiltrated Nicotiana benthamiana plants was not optimal. Consequently, the generation of Zera®-induced gp140 protein bodies was evaluated in a mammalian expression system. Stable HEK293 cell lines expressing Zera®-gp140 or gp140-Zera® were generated. A mixture of small PB-like structures was observed in cells expressing gp140-Zera®. However, no PB-like structures were seen in cells expressing Zera®-gp140. The immunogenicity of Zera®-gp140 and gp140-Zera® was evaluated by in rabbits. Binding and Tier 1A neutralising serum titres were higher for gp140-Zera® than for Zera®-gp140. Neither gp140-Zera® nor Zera®-gp140-specific sera neutralised a Tier 1B pseudovirus or the autologous Tier 2 CAP256SU pseudovirus, suggesting that Zera® might have compromised the structure of the Zera®-tagged gp140 proteins. The second approach investigated the two-component SpyCatcher/SpyTag technology. The stable HEK293 cell line expressing CAP256 gp140-SpyTag (gp140-ST) was generated, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in E. coli and purified by ultracentrifugation. The gp140-ST trimers and the SCAP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). SDS-PAGE, dynamic light scattering and negative stain electron microscopy indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs. The second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens and then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. These results demonstrate that careful selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of HIV-1 envelope trimers is an important consideration and that this could improve the effect of nanoparticle-displayed gp140.

No description available.

No description available.

The development of a safe, effective prophylactic HIV vaccine remains a major global health priority. Stabilized, soluble trimers that mimic the native functional HIV trimer have been developed that elicit strain-specific neutralizing HIV antibodies in animal models, and are currently being evaluated in several human clinical trials. Identifying whether multiple immunogens could be administered to facilitate the broadening of responses represents a pivotal challenge. In this thesis, we characterized the antibody response in individuals infected with multiple HIV strains to inform the development of polyvalent and sequential HIV vaccine regimens. We found that conventional approaches to detect HIV co- and superinfection are confounded by recombination. Therefore, we developed an automated, Bayesian approach to detect superinfection explicitly accounting for recombination. Using simulated and real sequence data, we demonstrated that this approach is sensitive, highly specific, and robust to recombination. Furthermore, analyzing previously published sequence datasets, we identified cases of superinfection that previously went undetected, indicating that superinfection occurs more frequently than previously estimated. We characterized the development of antibodies in five superinfected individuals identified in the CAPRISA 002 acute infection cohort. Specifically, we evaluated whether superinfection re-engaged cross-reactive memory B cells, promoting the development of cross-neutralizing antibodies. By comparing the breadth of the neutralizing antibody response in superinfected individuals to those that typically develop in singly infected individuals, we showed that HIV superinfection was not sufficient to broaden responses. By characterizing the kinetics and specificity of autologous neutralizing antibody responses, we show that responses to the superinfecting viruses failed to efficiently recruit neutralizing memory B cells. Instead, the secondary infection elicited strain-specific, de novo responses. This occurred even though the superinfecting viruses were relatively closely related (from the same subtype). To determine whether the co-exposure to diverse Env antigens favours the development of cross-neutralizing antibodies better than sequential exposure, we characterized the development of neutralizing antibodies in HIV co-infected individuals where several divergent viruses were transmitted prior to seroconversion. We identified three cases of co-infection that encompassed immunological exposure to: (i) two diverse, unlinked Envs, (ii) two related Envs with diversity uniformly distributed over the trimer, and (iii) two diverse but recombined Envs such that clusters of high homology were preserved in the presence of high diversity elsewhere. We found that, like superinfection, co-infection was not sufficient to broaden neutralizing antibody responses. Co-exposure to two HIV Env antigens did not necessarily produce additive or cross-neutralizing antibody responses, and in some cases was subject to immunological interference. This was most evident in the case of co-infection with two related Envs where diversity was uniformly distributed across the Env trimer; in this case neutralizing antibody responses to one variant arose to the near exclusion of responses to the other. However, in the case of co-exposure to diverse Envs but where the trimer apex was conserved in both variants through recombination, potent neutralization of both variants was evident. This was the co-infected participant who developed the broadest neutralizing antibody response, and we show that cross-neutralization was mediated, in part, by trimer apextargeting neutralizing antibodies. In conclusion, we find that HIV superinfection fails to efficiently recruit neutralizing memory B cells and, at best, results in additive nAb responses rather than a synergistic effect leading to cross-neutralization; a distinction that is highly relevant for vaccine design. While sequential immunizations with heterologous Env immunogens may be able to improve the potency of elicited responses, alone, they are unlikely to promote the development of bnAbs. Our observations from cases of co-infection suggests that cocktails of divergent stabilized Env trimers are unlikely to drive the development of cross-neutralizing antibodies, and may be subject to interference. However, the rational design of more similar immunogen cocktails where conserved epitopes are preserved across immunogens may be able to facilitate neutralizing antibodies to these targets, as seen in one individual. Thus, the use of related, stabilized Env trimers with diversity introduced in key regions together with strategies to reduce the immunogenicity of immunodominant, strain-specific epitopes may represent one path to a cross-neutralizing antibody response to multiple Envs within a cocktail.

Digitized using a Konica Minolta 211 PCL Scanner. 300dpi (OCR). / Thesis (MA (Psychology))--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The first objective of the present study was to determine whether the Theory of Planned Behaviour (TPB) could predict Willingness to Participate (WTP) in a future Phase III HIV vaccine trial among high risk adolescents in the Western Cape. The second objective was to determine whether the additional predictor variables of Self perceived risk of HIV infection, Knowledge of HIV vaccines and HIV vaccine trials, Attitudes toward HIV I AIDS and Health-promoting behaviours could further explain WTP in a future Phase III HIV vaccine trial among adolescents. A convenience sample of 224 adolescents attending secondary schools located in an African township on the Cape Flats was recruited for the present study. Hierarchical logistic regression analyses indicated that the TPB significantly improved the prediction of WTP in an HIV vaccine trial. Prediction success was 79.9%. Of all the predictor variables, only Subjective norms significantly predicted WTP in an HIV vaccine trial (OR = 1.19,95% C.L = 1.06-1.34). A second stepwise logistic regression analysis showed that Subjective norms (OR = 1.19, 95% c.I. = l.07-1.34) and Attitude towards participation in an HIV vaccine trial (OR = 1.32,95% C.L = 1.00-1.74) were significant predictors of WTP in an HIV vaccine trial. Prediction success was 80.4%. These findings provide support for the Theory of Reasoned Action (TRA) and suggest that psychosocial factors may play a role in WTP in a future Phase III HIV vaccine trial among adolescents. HIV vaccine trial preparedness programs targeting adolescents should aim to influence group norms positively and promote positive attitudes toward participation in a future Phase III HIV vaccine trial. / AFRIKAANSE OPSOMMING: Die eerste oogmerk van die huidige studie was om te bepaal of die teorie van beplande gedrag (TBG) die bereidwilligheid tot deelname (BTD) aan 'n toekomstige fase III-MIV-entstofproefneming onder hoe risiko adolessente in die Wes-Kaap kan voorspel. Die tweede oogmerk was om te bepaal of die bykomende voorspellingveranderlikes, naamlik selfwaargenome risiko vir MIV -besmetting, kennis van MIV -entstowwe en MIV -entstofproefnemings, houdings jeens MIV /Vigs en gesondheidsbevorderende gedrag BTD in 'n toekomstige fase III-MIV entstofproefneming kan verduidelik. 'n Geriefsmonster van 224 adolessente wat sekondere skole gelee in 'n dorpsgebied in Kaapstad, bywoon, is vir die huidige studie gewerf. Hierargiese logistiese regressie-analises toon dat die TBG die voorspelling van BTD aan 'n MIV -entstofproefneming aanmerklik verbeter het. Voorspellingsukses was 79.9%. Van al die voorspellingveranderlikes het slegs subjektiewe norme BTD aan 'n MIV -entstofproefneming beduidend voorspel (RK = 1.19, 95% VI = 1.06-1.34). 'n Tweede stapsgewyse logistiese regressie-analise toon dat subjektiewe norme (RK = 1.19, 95% VI = 1.07-1.34) en houding jeens deelname aan 'n MIV-entstofproefneming (RK = 1.32,95% VI = 1.00-1.74) beduidende voorspellers van BTD aan 'n MIV -entstofproefneming was. Voorspellingsukses was 80.4%. Hierdie bevindinge verleen steun aan die teorie van beredeneerde aksie (TBA) en doen aan die hand dat psigososiale faktore moontlik in die toekoms 'n rol in BTD aan 'n fase Ill-MIV-entstofproefneming onder adolessente kan speel. Programme wat op adolessente se gereedheid vir entstofproefnemings afgestem is, behoort te poog om groepnormne positief te beinvloed en positiewe houdings jeens deelname aan 'n toekomstige fase III -MIV -entstofproefneming te bevorder.

HIV in Karnataka, south India disproportionately burdens female sex workers (FSWs) and men who have sex with men (MSM). The best long-term strategy for managing the global HIV epidemic might involve a preventive vaccine; however, vaccine availability cannot guarantee its acceptability. An exploratory, cross-sectional study was conducted among frontline health service providers (HSPs) working with MSM/FSWs in relation to HIV-related health services in Karnataka. Face-to-face structured interviews were performed to better understand potential barriers/facilitators to acceptability/uptake of a future HIV vaccine among MSM/FSW communities. Descriptive analyses explored HSPs’ perceptions of vaccine acceptability/uptake and likelihood to recommend an HIV vaccine. Although HSPs mentioned numerous potential barriers to future HIV vaccine acceptability/uptake, most believed that MSM/FSWs would be willing to receive the vaccine to protect their health and avoid HIV. HSPs reported being very likely to recommend the vaccine, however young age of potential vaccine recipients negatively affected likelihood to recommend.

HIV in Karnataka, south India disproportionately burdens female sex workers (FSWs) and men who have sex with men (MSM). The best long-term strategy for managing the global HIV epidemic might involve a preventive vaccine; however, vaccine availability cannot guarantee its acceptability. An exploratory, cross-sectional study was conducted among frontline health service providers (HSPs) working with MSM/FSWs in relation to HIV-related health services in Karnataka. Face-to-face structured interviews were performed to better understand potential barriers/facilitators to acceptability/uptake of a future HIV vaccine among MSM/FSW communities. Descriptive analyses explored HSPs’ perceptions of vaccine acceptability/uptake and likelihood to recommend an HIV vaccine. Although HSPs mentioned numerous potential barriers to future HIV vaccine acceptability/uptake, most believed that MSM/FSWs would be willing to receive the vaccine to protect their health and avoid HIV. HSPs reported being very likely to recommend the vaccine, however young age of potential vaccine recipients negatively affected likelihood to recommend.

Malgré 30 ans de recherche aucun vaccin VIH n'a permis d'apporter une protection efficace et de manière stable. Au regard des échecs obtenus jusqu'à présent, de nouvelles formes vaccinales ont été développées. Parmi ces dernières les VLP présentent l'avantage notables d'être très immunogènes du fait de leur forme particulaire mimant les virus natifs, et sécuritaire puisque ne véhiculant pas de génome viral. Ce travail de thèse a pour objectif d’établir et d’évaluer une stratégie vaccinale utilisant ces VLP administrées par voies muqueuses dans le but d’initier une réponse humorale et cellulaire au niveau systémique et muqueux. Dans cette étude, nous avons montré que la voie muqueuse est indispensable pour l’induction d’une réponse locale forte. De plus, nos résultats révèlent que la forme particulaire de l’antigène est décisive dans la génération d’une immunité de qualité, générant une réponse TFH forte, une réponse cellulaire locale polyfonctionnelle ainsi qu’une réponse humorale forte et stable dans le temps, caractérisée par des anticorps de qualité.Cherchant à mieux caractériser la stratégie vaccinale établie, nous avons analysé les mécanismes de prise en charge des VLP et d’initiation de la réponse immunitaire après administration IN. Nous avons observé que de nombreuses cellules de l’immunité innée pulmonaire, notamment les macrophages alvéolaires et les neutrophiles, captaient massivement les VLP limitant alors la réponse TFH et potentiellement la réponse humorale qui en découle. Au final, ce travail de thèse aura permis de mettre en avant la voie d’immunisation muqueuse ainsi que la forme particulaire de l’antigène dans la mise en place d’un vaccin VIH. / Currently no HIV vaccine elicit full and stable protection against viral acquisition. In view of the failures until now, new vaccines strategies were developed. Among these, VLP have the advantage to be highly immunogenic because of their particulate structure mimicking native pathogens and safe because of the lack of viral genome.This thesis work aims to develop and evaluate a VLP-based vaccine strategy by mucosal administration in order to initiate systemic and local humoral and cellular responses. In this study, we showed that the mucosal administration is mandatory to generate a strong local immunity. Moreover, our results show that particular form of the antigen is crucial in the generation of the quality of the immune response, generating strong TFH response, polyfunctional T-cell responses in the mucosa and a strong and stable humoral response characterized by high-quality antibodies.We also investigated mechanisms involved in the generation of immune responses following IN VLP injections. We determined which cells take in charge VLP and their role in the followed immune responses. Our preliminary results show many innate immune cells in the lungs, such as alveolar macrophages and neutrophils, have an important role in the particles uptake, limiting TFH response and potentially the followed humoral response.Finally, this thesis work will show the determining role of the mucosal route of immunization and the particulate form antigen for the development of an HIV vaccine.

<p>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.</p>

<p>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.</p>