ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg 2017. / Background: Nasopharyngeal colonisation is a pre-requisite for developing bacterial respiratory and invasive disease. Immunisation of children with the pneumococcal conjugate vaccine (PCV) impacts upon colonising pneumococcal serotypes, which in turn could also affect the biome of the nasopharynx in relation to colonisation by other bacteria. Due to limitations in standard culture methods, the association between PCV-immunisation and bacterial carriage density is still unclear, including among HIV-infected children. In this study we aimed to evaluate the effect of infant vaccination with the 7-valent PCV (PCV7) on vaccine-serogroup colonisation in order to determine whether the increase in non-vaccine serotype (NVT) colonisation was due to unmasking of previously low density colonising serotypes or increase in acquisition of NVT. Also, we evaluated the association between PCV7 immunisation and HIV-infection on the prevalence density of nasopharyngeal colonisation by other common potentially pathogenic bacteria. Methods: A multiplex real-time qPCR assay was set up to detect 44 common pneumococcal serotypes and 5 bacterial pathogens including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pyogenes. All assays were optimised according to MIQE guidelines and their ability to detect multiple pneumococcal serotype/group and bacteria in archived nasopharyngeal swabs were evaluated. The multiplex qPCR assays were then used to evaluate vaccine-serotype, non-vaccine serotype and bacterial nasopharyngeal colonisation in achieved swabs of PCV7-vaccinated (at 6, 10 and 14 weeks of age) and PCV-unvaccinated African children at 9 and 15-16 months of age, prior to routine vaccination of children with PCV through the public immunisation program. In order to address the limitations of the qPCR assays, a nanofluidic real-time PCR assay was developed to simultaneously detect 53 pneumococcal serotypes, 6 serotypes of H. influenzae and 11 bacterial pathogens. Further, all assays were optimised and evaluated according to the MIQE guidelines and findings from Fluidigm and traditional qPCR assays were compared. Lastly, Fluidigm was used to evaluate the association of HIV-infection on the prevalence and density of nasopharyngeal colonisation at 9 and 16 months of age by common, potentially pathogenic bacteria including PCV7 pneumococcal serotypes, non-PCV7 serotypes, Haemophilus influenzae, non-typable Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Streptococcus pyogenes, Neisseria meningitidis, Neisseria lactamica, Bordetella pertussis, Bordetella parapertusis, Bordetella bronchiseptica and Bordetella holmesii in achieved nasophartngeal swabs collected from PCV7-vacciniated HIV-infected and HIV-uninfected children. Results: Molecular qPCR was more sensitive than culture in detecting multiple concurrent colonising pneumococcal serotypes as well as other common nasopharyngeal colonisers, with the majority of additional isolates detected by qPCR having a low carriage density (<104 CFU/ml). Further, qPCR identified a lower prevalence of PCV7-serotype colonisation among PCV7-vaccinated compared to PCV-unvaccinated children at 9 and 16 months of age [adjusted Odds Ratio (aOR): 0.37; 95% CI; 0.19-0.7 and 0.41; 95% CI; 0.26-0.63, respectively]; and an increase in NVT-serotype [aOR: 1.88; 95% CI; 1.02-3.48 and 2.2; 95% CI; 1.18-4.1] colonisation respectively. The increase in NVT carriage among PCV7-vaccinees was driven by serotype 19A, which increased by 53.4% (p=0.021) and 70.7% (p<0.001) at 9 and 16 months of age respectively. Further, 19A had a higher density of colonisation in PCV7-vaccinated groups compared to PCV-unvaccinated groups and was more likely to be identified as a primary than non-primary isolate in PCV7-vaccinated children alone. PCV immunisation was also associated with an increased prevalence of H. influenzae at 9 months (55.8% vs. 66.3%, p<0.001) and 16 months (72% vs. 62%, p=0.017) of age, while a temporary increase in the carriage prevalence of S. aureus was found in PCV7-vaccinated (18.9%) compared to PCV-unvaccinated children (11.1%, aOR 2.1; 95% CI 1.0-1.4; p=0.049) at 9 months of age only. The density of pneumococcus (4.68 vs. 4.28 CFU/ml; p=0.007), H. influenzae (3.86 vs. 4.34 CFU/ml; p=0.008), M. catarrhalis (2.98 vs. 3.52 CFU/ml; p<0.001) and S. aureus (3.06 vs. 4.02 CFU/ml; p=0.02) were also higher among PCV7-vaccinated compared to PCV-unvaccinated children at 9 months age, although this difference diminished with increasing age. There was excellent concordance between the qPCR and Fluidigm for carriage prevalence and density of the majority of assays, with Fluidigm identifying an additional 7 pneumococcal serotypes and 11 bacterial species above those detected by qPCR. Further, discordant results between the two PCR methods were strongly associated with a low carriage density (<102 CFU/ml). Using molecular Fluidigm, a lower carriage prevalence of overall pneumococci (58.6% vs. 69.9%; p=0.02), non-vaccine serotypes (27.8% vs. 40%; p=0.047) and H. influenzae (64.2% vs. 42.3%; p=0.01) was identified in HIV-infected children compared to HIV-uninfected children who were immunised with PCV7 at 9 months of age. No difference in the carriage prevalence of overall pneumococci was however found at 16 months of age (p=0.20), although the carriage prevalence of non-vaccine serotypes (50.9% vs. 60.4%; p=0.049) and H. influenzae (56% vs. 73.4%; p=0.02) was lower in HIV-infected children at 16 months of age. In addition, the density of overall pneumococcus was found to be higher in HIV-infected children (4.81 vs. 4.44 CFU/ml; p=0.014), despite the lower carriage prevalence at 9 months of age, which was driven by a higher density of vaccine serotypes/serogroups (4.21 vs. 3.72 CFU/ml; p=0.04). By 16 months of age, there was no difference in density of pneumococcal colonisation between the HIV-infected and HIV-uninfected children (p=0.89). No difference in the density of H. influenzae was found between HIV-infected and HIV-uninfected infants at 9 months of age (p=0.08); however, by 16 months of age, HIV-uninfected children had a higher density of overall H. influenzae colonisation (4.95 vs. 4.32 CFU/ml; p<0.001), which was largely due to the higher carriage density of NThinf in HIV-uninfected children (5.0 vs. 4.23 CFU/ml; p<0.001). Conclusion: Molecular qPCR assays were shown to be a promising alternative to WHO recommended culture in that multiple pneumococcal serotypes and other bacterial pathogens could be simultaneously detected as well as the bacterial load of each colonising bacteria quantified. The mechanism behind the vaccine effect was shown to be a combination of both serotype replacement and unmasking; however, the reduction in PCV7-serotype colonisation impacted on colonisation prevalence and density of other bacterial species of the nasopharynx and the clinical relevance of this needs further exploration in relation to mucosal and invasive disease outcomes, as well as for higher valence vaccines. While the higher carriage density of overall pneumococcus in HIV-infected children, despite the lower carriage prevalence might explain the higher invasive disease burden in HIV-infected compared to HIV-uninfected children even in the era of antiretroviral therapy treatment and PCV immunisation, future studies are required to provide clarity. Nevertheless, the findings from this thesis highlight the importance of continued surveillance of the circulation of pneumococcal serotypes as well as other bacterial pathogens especially in a population with a high burden of HIV-1 infection. / MT2017

The efficacy of some vaccines is enhanced by the presence of adjuvants added to their formulations or, in the case of live attenuated or killed whole cell vaccines, because of their endogenous adjuvant activity. The immune system responds robustly to these endogenous adjuvants, which includes Pathogen Associated Molecular Patterns, which stimulate innate immune responses through Pattern Recognition Receptors, such as TOLL-like receptors (TLRs). The development of most vaccine adjuvants has occurred despite little understanding of their overall mechanisms of immune enhancement. We hypothesized that TLR-dependent adjuvant activities are mediated through TLR stimulation of antigen presenting cells (APCs), and each APC type may play a unique role in the immune-stimulating ability of these adjuvants, including effects on downstream T cell stimulation. We used a mouse model where TLR/MyD88 signaling is prevented in specific APC types, in vivo, using loxP/cre recombinase transgenic mice (B cells, dendritic cells and macrophages) to investigate its role in vaccine adjuvant activity. We found that intact MyD88 signaling is essential, separately, in all three APC types for optimal TLR-ligand based adjuvant (PorB, CpG, MPLA), but not for TLR-independent (Alum, MF59) adjuvant activity. However, the immune responses were reduced to the greatest extent in mice with macrophage specific MyD88 deletion (Mac-MyD88-/-). We demonstrated that TLR-dependent adjuvants are potent inducers of germinal center (GCs) formation needed for an effective and robust immune response. Interestingly, GCs are nearly absent in Mac-MyD88-/- mice upon immunization with TLR-dependent adjuvants, but not with TLR-independent adjuvants. Further investigations revealed a significant impairment in T cell cytokines important for GC formation in Mac-MyD88-/- mice when immunized with TLR-dependent adjuvants. Through these studies we discovered that vaccine formulated with PorB/OVA induced a robust and diverse T cell response including highly functional OVA-CD4 and CD8 T cells. These CD8 T cells are protective and significantly reduced the bacterial burden and increased survival in a Listeria mouse infection model. Our findings reveal that PorB has broad adjuvant activity, signaling through all three APC types, inducing strong and diverse humoral and cellular responses. These insights will allow for a more intelligent use of adjuvants in future vaccine development.

PURPOSE: The purpose of this study was to investigate the potential relationship between MMR vaccination coverage and measles incidence in the US, as well as to examine the demographic characteristics and socio-economic status of unvaccinated individuals to determine if there are certain sub-populations who are routinely not receiving the MMR vaccine. METHODS/PROCEDURES: This retrospective cohort study determined the MMR vaccination coverage per year and compared it to the measles incidence for the same year. RESULTS: Results from this study suggest that regional differences in MMR vaccination rates spanning across multiple sub-populations are associated with the increasing measles incidence in the US. CONCLUSIONS: The correlations between MMR vaccination coverage and measles incidence in the US should be investigated further to determine what specific programs can be put in place to increase MMR vaccination rates state-wide and among vulnerable sub-populations.

Development of a broadly cross-protective capsular group B meningococcal (MenB) vaccine has been hampered by poor capsular immunogenicity and often diverse and poorly cross-protective subcapsular antigens. The MenB MC58 strain genome has facilitated the discovery of novel, relatively conserved vaccine candidates. The four-component MenB (4CMenB) investigational vaccine contains factor H-binding protein (fHbp; variant 1), neisserial heparin-binding antigen (NHBA), Neisserial adhesin A (NadA) and PorA P1.4-containing outer membrane vesicles. The latter are known to elicit protection against homologous strains. Clinical trials have demonstrated protective responses in infants and adults against isolates expressing homologous PorA or fHbp (subvariant 1.P1), or heterologous NadA (variant 2). Cross-protective responses have also been demonstrated in adults and, to a lesser extent, infants, against isolates expressing heterologous fHbp variant 1 subvariants. The contribution of NHBA is still poorly understood. MenB currently accounts for 87% of invasive meningococcal disease in England and Wales. The proportion of disease due to the ST-269 clonal complex (cc269) peaked at 45.6% in 2006 and is currently approximately 24.2%. The aims of this study were (i) to genotypically assess potential 4CMenB coverage against recent English and Welsh invasive disease isolates and, specifically, cc269 isolates from England and Wales and other countries, (ii) to compare phenotypic expression levels of the 4CMenB antigens (excluding PorA) among typical cc269 isolates, and (iii) to assess 4CMenB responses against typical cc269 isolates among healthy adults administered 4CMenB.Full length alleles for fHbp variant 1, NHBA and NadA variants 1, 2 and 3 were present in 64.6%, >99% and 7.1%, respectively, of English and Welsh invasive disease isolates from 2007/8. Between 67.5% and >99% (adults) or 25.7% and 43.5% (infants) of the isolates were predicted to be covered by 4CMenB. cc269 comprised two antigenically distinct lineages (clusters) centred around ST-269 and ST-275, respectively. These accounted for 57% and 40% of cc269 in 2007/8. Both clusters effectively lacked nadA and PorA P1.4. The predominant fHbp;NHBA profiles represented by the respective clusters were 1.P15;P0021 and (1.P13 or 2.P19);P0017. Between 77.4% and 100% (adults) or 2.2% and 27.1% (infants) of cc269 isolates from 2007/8 were predicted to be covered by 4CMenB. Estimates for infants were conservative due to e.g. the exclusion of NHBA. Serum bactericidal antibody (SBA) analyses targeting typical fHbp variant 1-expressing cc269 strains, indicated high levels of coverage among adults administered 4CMenB. Notable differences among genotypically matched isolates e.g. in terms of SBA geometric mean titres, were not reflected in the relative fHbp and NHBA expression levels. Such differences may lead to conflicting estimates of coverage in infant populations. Whilst these are investigated further it seems prudent to use typical isolates giving mid-range responses when assessing SBA, and therefore protection, among infants. Potential 4CMenB coverage of cc269 and the broader meningococcal population in England and Wales was high among adults and encouraging among infants when compared to that of existing MenB vaccines.

Pertussis, also known as whooping cough, is a vaccine-preventable disease that is on the rise in the United States, a trend which has been attributed to vaccination exemption. Indeed, the pertussis outbreak that occurred in February, 2012 in Ocean County, New Jersey was associated with vaccine exemption. Considering that pertussis is deadly to young children, it is important to understand why this disease rate is on the rise. The research questions were focused on whether a relationship existed between pertussis status (no, yes) and exemption status (no, yes), sex (male, female), and county type (Middlesex, Ocean, or Other), using a theoretical foundation of eco-social theory. The methodology used in the study was a retrospective case-control design. Archival data were collected on residents of Ocean County New Jersey; Middlesex County, New Jersey; and New Jersey as a whole using nonprobability purposive sampling (n = 63,000). A power analysis was conducted for sample size and chi square test of association was performed for data analysis. The results supported the hypotheses that a significant difference existed in the prevalence of pertussis between Ocean County, Middlesex County, and all other counties in New Jersey. The data showed that the odds of being afflicted with pertussis for those residents of Ocean County was greater than it was for those residents living in other counties in New Jersey, though sex was not found to be a significant variable. This study can promote social change by providing public health officials important knowledge about the nature of the outbreak, supporting public health practices designed for the population at risk. Resource allocation can be more specifically targeted to enhance disease reduction by creating programs designed to populations presenting the greatest risk of disease spread.

n/a

全球疫苗市場85%是五大疫苗廠Sanofi Pasteur、MSD、GSK、Pfizer及Novartis所寡占。跟隨著研發技術發展、持續的疫苗政策、疾病地區化、公衛防治的提升，極大的市場需求，加速疫苗上市持續帶動疫苗產業成長。疫苗的預防接種是防治傳染病，最直接及最具經濟效益的投資，也是國家防疫能力的展現。本研究主要目的為彙整全球疫苗產業總體產業環境現況，探討影響全球疫苗產業發展之關鍵因素及我國疫苗政策發展。 本研究的主要研究方法，採用檔案分析與個案研究法，前者包括次級資料收集及彙整，後者包括疫苗政策形成、實務推動之相關人士的訪談。 本研究的結論 (1)寡占的疫苗產業及疫苗產業地區化的發展趨勢，是影響未來全球疫苗產業及我國疫苗產業發展之關鍵因素。(2)財源籌集制度會影響我國疫苗政策的擬定。(3)疫苗受害救濟機制與持續教育是疫苗政策推動必要之輔助制度。(4)提升疫苗產業成為國家經濟產業，是突破法規鴻溝及研發速度的產業扶植手段之一。 / Over 85% of vaccine market was controlled by top 5 vaccine manufactures: Sanofi Pasteur, MSD, GSK, Pfizer, and Novartis. Nowadays, research and development technology are advancing; vaccine policies are continued; diseases are regionalized; public health control and prevention are increased. Thus, an extreme market demand resulted and expedited the introduction of vaccines to the markets, driving the ongoing growth of the vaccine market. Vaccination is the most direct and cost-effective investment against infectious diseases and also the demonstration of a country’s ability to against the disease outbreak. The purpose of this study was to summarize the trend of the vaccine market and explore key factors affecting the development of the global vaccine market and vaccine policy in Taiwan. This study was carried out mainly through documentation method and in depth interview case study. Documentation method included the collection and summary of secondary data while the interview case study was to interview the experts who were involved in the formation of vaccine policy and promotion. Conclusions of this study are：(1)The top 5 vaccine manufactures and vaccine industry regionalization are the key factors affecting the development of the vaccine market across the world as well as Taiwan in the future；(2)The vaccine financing affects the formulation of vaccine policy in Taiwan；(3)The vaccine injury compensation program and continuing education as auxiliary systems are necessary mechanisms for the vaccine policy promotion；and (4)Promoting the vaccine industry to the national economic industry level is one of the ways to amend regulatory gaps and advance R&D speed for the benefit of the industry.

Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft-versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant-related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients. The aim of the work described in this thesis has been to implement a strategy to transplant a patient using a HLA haploidentical donor. The strategy is to try to improve DFS that could be applied both in the autologous or allogeneic context: first, by using nonspecific immune manipulation post transplant and then, by developing specific strategies directed against leukemia antigens. Particularly in the allogeneic situation, the aim was to increase the GVL effect without inducing or aggravating the deleterious GVHD. The first part of this thesis described our own clinical results, consisting of three consecutive phase I/II studies, in which we tried to determine the feasibility of giving prophylactic donor lymphocyte infusions (DLI) post transplant and the effect of replacing granulocyte colony-stimulating factor (G-CSF), typically used to speed up neutrophil recovery, with granulocyte macrophage colony-stimulating factor (GM-CSF), which is known for its immunomodulatory properties. The slow immune reconstitution in haploidentical transplant is chiefly responsible for the high incidence of early lethal viral and fungal infections, and most probably for early relapses; therefore, we sought to accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. Thus, we have studied the impact of post transplant growth factor administration and of unselected DLI in haploidentical transplant. We have also implemented, in our center, anti-cytomegalovirus (CMV) specific T cell generation and infusion to improve anti-CMV immune reconstitution. Since then, our results have been pooled in a multi-center analysis performed by the European Bone Marrow Transplantation group (EBMT) allowing us to compare our results with those of the entire group. We have also participated in the design of an ongoing study aimed at selectively depleting the graft from alloreactive T cells, and improving post transplant T cell add-backs. In our attempts to generate and expand ex vivo lymphocytes (directed against pathogens (CMV) and leukemia-associated antigens, Wilms' tumor gene 1 (WT1) and to use them in vivo, we found inconsistent results (in the case of WT1) using classical clinical grade dendritic cells (DC) generated and matured in bags, as was the case for the majority of the teams worldwide. This led us to question the full functionality of these DC and we undertook a thorough comparative analysis of DC generated and differentiated in bags and in plates (typical for most pre-clinical studies). This analysis showed us that one cannot transpose pre-clinical studies (using culture plates) directly to clinical protocols (generally using clinical grade culture bags) and that DC generated in bags are functionally deficient. We learned that, if we want to use a DC vaccine to improve the GVL effect in haploidentical transplant, we will have to be careful about the technique by which they are generated. To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to manipulate them is critical in the development of new effective immunotherapeutic clinical trials. This is why we currently focus on how to obtain effective in vivo anti-leukemia immune reactions using an ex-vivo manipulated product to trigger the immunotherapeutic response. More specifically, we are analyzing the impact of regulatory T cell (Tregs) depletion and function for an adequate anti-leukemic immune response. This pre-clinical work aims at improving the outcome of leukemia patients who have relapsed and been put back into second remission and at decreasing the RR after HSCT, especially in the field of haploidentical transplantation. In conclusion, haploidentical transplantation has become a valuable tool. The results are at least similar to those obtained using MUD when performed in the same group of patients. Specific immunomodulation post transplant can affect events such as GVHD and GVL, but clinically we are still at the level of nonspecific manipulations. It is our hope that ongoing pre-clinical work will enable us to perform specific anti-pathogen and anti-leukemia immune manipulation that will favorably influence the patient outcome. / Dans la majorité des situations, le système immunitaire autologue est incapable d’éradiquer les cellules leucémiques résiduelles qui échappent à la radiothérapie et à la chimiothérapie, cependant un équilibre peut s’établir entre les cellules leucémiques et immunitaires aboutissant à une rémission pouvant durer plusieurs mois ou années. Si cet équilibre se rompt, une rechute clinique peut se déclarer. Dans ce contexte, il est prouvé que la greffe allogénique de cellules souches hématopoïétiques est le moyen le plus efficace de renforcer les réactions immunitaires contre la leucémie par la réaction du greffon contre la leucémie et ainsi d’obtenir une éradication définitive de la maladie résiduelle chez un nombre significatif de patients. En effet, le concept global de l’allogreffe de cellules souches hématopoïétiques a évolué du concept de transplantation d’organe (remplacement d’un organe malade par un nouvel organe sain) vers celui de créer une extraordinaire plateforme d’immunothérapie à travers laquelle le système immunitaire du donneur contribue à l’éradication des cellules leucémiques persistantes. Donc, la problématique reste celle de trouver les meilleures modalités d’immunomodulation pour achever une prise du greffon, un effet anti-leucémique puissant du greffon, et l’absence ou un minimum d’effet du greffon contre l’hôte. Différentes stratégies existent pour atteindre cet objectif, comme l’utilisation de cytokines pour moduler la reconstitution immunitaire, des déplétions cellulaires globales ou spécifiques du greffon et l’infusion de cellules immunes «globales» ou spécifiques du donneur après greffe. Ces stratégies sont encore largement à l’étude. Néanmoins, la persistance d’un taux de rechute élevé observé chez les patients leucémiques, après allogreffe reste la cause principale de décès, avant celle liée à la toxicité de la greffe. De plus, étant donné que seulement environ 40 à 70% (dépendant de l’origine ethnique) des patients avec une hémopathie à haut risque, éligibles pour une greffe allogénique, ont un donneur familial ou non familial complètement HLA compatible, des efforts importants ont été développés pour rendre faisable l’utilisation de donneurs familiaux alternatifs, haploidentiques. L’avantage de cette approche est l’accès immédiat à un donneur pour quasiment tous les patients. Le but du travail décrit dans cette thèse a été l’implémentation d’une stratégie d’allogreffe utilisant un donneur haploidentique. Le travail vise également à développer de façon plus large des stratégies qui peuvent améliorer le taux de survie sans rechute, non seulement dans le contexte des greffes haploidentiques, mais également dans le cadre des greffes allogéniques en général, ainsi que dans les situations autologues : premièrement, par la manipulation immunitaire non spécifique après greffe et ensuite par le développement de stratégies spécifiques dirigées contre des antigènes leucémiques. En particulier dans la situation allogénique, le but a été d’augmenter l’effet du greffon contre la leucémie sans induire ou aggraver l’effet délétère du greffon contre l’hôte. La première partie de la thèse décrit les résultats cliniques de notre propre protocole de greffe haploidentique, qui a consisté en trois études consécutives de phase I/II. Dans ces études, nous avons voulu déterminer la faisabilité de réaliser des infusions prophylactiques de lymphocytes du donneur après transplantation, et l’impact du remplacement du « granulocyte colony-stimulating factor » (G-CSF), largement utilisé pour permettre une récupération en polynucléaires neutrophiles plus rapide, par du « granulocyte-macrophage colony-stimulating factor » (GM-CSF), lequel est connu pour ses propriétés immunomodulatrices différentes. La reconstitution immunitaire très lente après greffe haploidentique est majoritairement responsable de l’incidence élevée de décès par infections virales et fungiques précoces, et très probablement des rechutes précoces. C’est pourquoi nous avons cherché à accélérer et à renforcer la reconstitution immunitaire post-greffe sans augmenter la fréquence de réaction du greffon contre l’hôte. Nous avons donc étudié l’impact de l’administration de facteurs de croissance et l’infusion de lymphocytes non sélectionnés du donneur en post greffe haploidentique. Nous avons également implémenté dans notre centre, la génération et l’infusion de lymphocytes T spécifiques anti-cytomégalovirus (CMV) afin d’améliorer la reconstitution immunitaire anti-CMV. D’autre part, nos résultats ont été regroupés dans une étude multicentrique menée par le groupe européen de transplantation de moelle osseuse (EBMT), ce qui nous a permis de comparer nos résultats avec ceux de l’entièreté du groupe. Nous avons parallèlement participé à la conception d’une étude actuellement en cours ayant pour but d’améliorer la reconstitution immunitaire après greffe par la déplétion sélective du greffon en lymphocytes T alloréactifs et par l’infusion après greffe de lymphocytes T du donneur également sélectivement déplétés en lymphocytes T alloréactifs. Afin d’optimaliser l’effet anti-leucémique du système immunitaire, nous avons débuté un protocole de vaccination par cellules dendritiques (DCs). Ces cellules dendritiques étaient chargées en lysat de blastes leucémiques dans le cas de patients présentant au diagnostic une leucémie aigue surexprimant l’oncogène 1 de la tumeur de Wilms (WT1). Néanmoins dans nos travaux de génération et d’expansion ex-vivo de lymphocytes T spécifiques de l’antigène WT1, utilisant les DCs de grade clinique, générées et maturées en poches, nous avons rencontré des résultats inconsistants, comme c’était le cas dans la majorité des protocoles cliniques internationaux de vaccination. Nous nous sommes alors posé la question de la fonctionnalité globale de ces cellules et nous avons entrepris une analyse comparative poussée des DCs générées et différenciées en poches ou en plaques. Les DCs générées en plaques sont celles utilisées dans la plupart des travaux précliniques. Cette analyse nous a montré que l’on ne pouvait pas directement transposer les résultats précliniques basés sur des DCs générées en plaques dans des protocoles cliniques basés sur des DCs générées en poches, car ces dernières présentent des déficits fonctionnels importants. Nous avons appris que si l’on voulait utiliser un vaccin à base de cellules dendritiques pour améliorer l’effet du greffon contre la leucémie dans les greffes allogéniques, nous devions être très attentifs quant au protocole utilisé pour la génération de ces vaccins cellulaires. Pour améliorer les approches immunothérapeutiques, la connaissance des mécanismes qui établissent la tolérance tumorale et des façons de manipuler ceux-ci, est critique dans le développement de nouveaux protocoles efficaces. C’est pourquoi nous nous concentrons actuellement sur les conditions nécessaires à l’obtention in vivo d’une réaction immune anti-leucémique efficace lors de l’utilisation d’un produit cellulaire manipulé ex vivo. Plus spécifiquement, nous analysons l’impact de la déplétion en lymphocytes T régulateurs (Tregs) sur la réponse anti-leucémique. Ce travail préclinique a pour but d’améliorer le devenir de patients leucémiques qui ont rechutés et ont été mis en seconde rémission, ainsi que de diminuer le taux de rechute après allogreffe, spécifiquement après greffe haploidentique. En conclusion, la transplantation haploidentique est actuellement un outil précieux pour de nombreux patients. Les résultats sont au minimum similaires à ceux qui sont obtenus par les greffes non-familiales HLA identiques lorsqu’elles sont pratiquées dans les mêmes groupes de patients. L’immunomodulation spécifique après greffe peut affecter des événements comme la réaction du greffon contre l’hôte et la réaction du greffon contre la leucémie, mais en pratique clinique nous en sommes encore au niveau de la manipulation aspécifique. Nous espérons que les travaux précliniques actuels vont nous permettre d’appliquer des stratégies spécifiques et d’obtenir une manipulation immune anti-leucémique qui aura une influence favorable significative sur le devenir des patients.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.