Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates.

UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

Role of Inflammatory Monocytes in Vaccine-Induced Reduction of Helicobacter felis Infection.

Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. In this study, we evaluated the role of inflammatory monocytes in the vaccine-induced reduction of Helicobacter felis infection. We first showed by using flow cytometric analysis that Ly6C(low) major histocompatibility complex class II-positive chemokine receptor type 2 (CCR2)-positive CD64(+) inflammatory monocytes accumulate in the stomach mucosa during the vaccine-induced reduction of H. felis infection. To determine whether inflammatory monocytes played a role in the protection, these cells were depleted with anti-CCR2 depleting antibodies. Indeed, depletion of inflammatory monocytes was associated with an impaired vaccine-induced reduction of H. felis infection on day 5 postinfection. To determine whether inflammatory monocytes had a direct or indirect role, we studied their antimicrobial activities. We observed that inflammatory monocytes produced tumor necrosis factor alpha and inducible nitric oxide synthase (iNOS), two major antimicrobial factors. Lastly, by using a Helicobacter in vitro killing assay, we showed that mouse inflammatory monocytes and activated human monocytes killed H. pylori in an iNOS-dependent manner. Collectively, these data show that inflammatory monocytes play a direct role in the immunization-induced reduction of H. felis infection from the gastric mucosa.

Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Immune correlates of vaccine protection against HIV-1 acquisition.

The partial efficacy reported in the RV144 HIV vaccine trial in 2009 has driven the HIV vaccine field to define correlates of risk associated with HIV-1 acquisition and connect these functionally to preventing HIV infection. Immunological correlates, mainly including CD4(+) T cell responses to the HIV envelope and Fc-mediated antibody effector function, have been connected to reduced acquisition. These immunological correlates place immunological and genetic pressure on the virus. Indeed, antibodies directed at conserved regions of the V1V2 loop and antibodies that mediate antibody-dependent cellular cytotoxicity to HIV envelope in the absence of inhibiting serum immunoglobulin A antibodies correlated with decreased HIV risk. More recently, researchers have expanded their search with nonhuman primate studies using vaccine regimens that differ from that used in RV144; these studies indicate that non-neutralizing antibodies are associated with protection from experimental lentivirus challenge as well. These immunological correlates have provided the basis for the design of a next generation of vaccine regimens to improve upon the qualitative and quantitative degree of magnitude of these immune responses on HIV acquisition.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Health care workers' influenza vaccination: motivations and mandatory mask policy

BACKGROUND: Vaccination of health care workers (HCW) against seasonal influenza (SI) is recommended but vaccination rate rarely reach >30%. Vaccination coverage against 2009 pandemic influenza (PI) was 52% in our hospital, whilst a new policy requiring unvaccinated HCW to wear a mask during patient care duties was enforced. AIMS: To investigate the determinants of this higher vaccination acceptance for PI and to look for an association with the new mask-wearing policy. METHODS: A retrospective cohort study, involving HCW of three critical departments of a 1023-bed, tertiary-care university hospital in Switzerland. Self-reported 2009-10 SI and 2009 PI vaccination statuses, reasons and demographic data were collected through a literature-based questionnaire. Descriptive statistics, uni- and multivariate analyses were then performed. RESULTS: There were 472 respondents with a response rate of 54%. Self-reported vaccination acceptance was 64% for PI and 53% for SI. PI vaccination acceptance was associated with being vaccinated against SI (OR 9.5; 95% CI 5.5-16.4), being a physician (OR 7.7; 95% CI 3.1-19.1) and feeling uncomfortable wearing a mask (OR 1.7; 95% CI 1.0-2.8). Main motives for refusing vaccination were: preference for wearing a surgical mask (80% for PI, not applicable for SI) and concerns about vaccine safety (64%, 50%) and efficacy (44%, 35%). CONCLUSIONS: The new mask-wearing policy was a motivation for vaccination but also offered an alternative to non-compliant HCW. Concerns about vaccine safety and efficiency and self-interest of health care workers are still main determinants for influenza vaccination acceptance. Better incentives are needed to encourage vaccination amongst non-physician HCW.

Les vaccins contre le zona : efficacité, sécurité, et rapport coût/bénéfices [Vaccines against Herpes zoster: Effectiveness, safety, and cost/benefit ratio].

CONTEXT: A vaccination against herpes zoster and its complication is available in France since June 2015. Its exact benefit for public health is still controversial and its level of protection is not optimal. All those reasons seem to suggest a low acceptation rate from general practitioners. OBJECTIVE: To evaluate the effectiveness, the safety, and the cost/benefit ratio of the vaccination against herpes zoster in people aged 50 year or over. DOCUMENTARY SOURCE: Systematic review in Medline and PubMed with research by key words: "herpes zoster vaccine", "zoster vaccine" and "post herpetic neuralgia vaccine". SELECTION OF STUDIES: Randomized and observational studies published in English and French language have been selected by two readers. RESULTS: On 1886 articles identified, 62 studies were included in this systematic review of which 21 randomized trials, 21 observational studies, and 17 medico-economic studies concerned the unadjuvanted vaccine. Considered studies showed an effectiveness of 50% against herpes zoster and 60% on post-herpetic neuralgia incidence of the unadjuvanted vaccine. Five randomized controlled studies were identified for the adjuvanted vaccine. The overall effectiveness of this vaccine was > 90% whatever the age of subjects including those over age 70 and 80. The medico-economic studies conducted in many countries have shown that vaccine policies were beneficial in individuals aged 60 years or over. LIMITATION OF THE WORK: Most of data of effectiveness, and tolerance result from 2 large controlled studies only (SPS and ZEST) for the unadjuvanted vaccine and only one for the adjuvanted vaccine. CONCLUSION: Despite controversy and few uncertainties, the vaccine significantly reduces herpes zoster and its complication incidence. In terms of public health objectives, it reduces the burden of the disease and has a positive medico-economic impact. Preliminary data concerning the adjuvanted vaccine, whilst very promising, are still too limited. Up to now, no group of people with particularly high risk of herpes zoster-related complication who will beneficiate the most of the vaccination has been identified yet and only an age criteria has been considered for the recommendation.

Influence of PTPN2 and pre-existing immunity on the generation of effector and memory CD8 T cells

Notre système immunitaire joue un rôle important pour la protection envers les maladies infectieuses. Au cours d'une réponse à une infection primaire, des cellules B et des cellules T spécifiques, dirigées contre le pathogène en question, sont générées et certaines d'entre elles deviennent des cellules dites mémoires. Leur fonction est de nous protéger contre une nouvelle infection avec le même pathogène, une infection secondaire. Dans certaines situations, comme c'est par exemple le cas avec la grippe, les pathogènes ne sont pas toujours complètement identiques et les cellules mémoires ne sont pas à même d'assurer leur rôle protecteur et d'empêcher une réinfection. Pourtant, on ne sait à l'heure actuelle que très peu comment une immunité acquise, mais non protectrice, influence le développement d'une réponse immunitaire ultérieure. Dans la première partie de cette thèse, nous avons étudié comment les cellules T mémoires cytotoxiques altèrent la réponse de cellules T cytotoxiques nouvellement induites. Au cours d'une réaction immunitaire dirigée contre une infection primaire, un vaste répertoire de lymphocytes T est créé, constitué de cellules T possédant divers degrés d'affinité pour le pathogène. Lors d'une infection secondaire, seules les cellules T ayant une forte affinité pour le pathogène participent à la réponse. Nous avons pu démontrer que ce phénomène de restriction du répertoire des cellules T est principalement causé par les cellules T mémoires qui sont à même de reconnaître un antigène pathogénique présent dans les deux infections. Dans un deuxième projet, nous avons étudié comment l'absence de PTPN2 influence la réponse des cellules T. Chez l'homme, une mutation dans le gène de PTPN2 est associée à des maladies auto-immunes et résulte en une activité réduite de cette phosphatase dans les lymphocytes T. Nous avons montré que la baisse d'activité de la phosphatase PTNP2 conduit à une meilleure expansion des cellules T ayant une qualité comparable à des cellules T auto-antigène spécifiques. De plus, nous avons observé que la survie de ces cellules T effectues ayant une phosphatase diminuée est nettement améliorée. Cela peut conduire à une réponse immunitaire plus efficace ou, éventuellement, à une pathologie auto-immune plus grave. En outre, nos résultats montrent qu'en manipulant l'activité de cette phosphatase, il est possible d'augmenter l'efficacité du transfert des cellules T dans un hôte receveur. Un tel transfert de cellules T est pratiqué chez des patients atteints de tumeurs. Nos travaux suggèrent que la manipulation de la phosphatase PTPN2 pourrait donc représenter une approche thérapeutique novatrice et prometteuse. -- Notre système immunitaire joue un rôle important pour la protection contre les maladies. Les cellules T CD8+ ont une importance primordiale pour le contrôle d'infections primaires causées par des virus ou bactéries, mais également contre certaines tumeurs. Par conséquent, mieux comprendre les exigences nécessaires à l'induction de bonnes réponses des cellules T CD8 pourrait nous permettre de construire des vaccins contre les pathogènes contre lesquels nous n'avons pour l'instant pas de vaccins mais aussi d'améliorer les réactions immunitaires dirigées anti-tumorales. Dans la première partie de cette thèse, nous avons étudié l'influence qu'une immunité préexistante a sur la réponse des cellules T CD8. Nous sommes souvent exposés à des pathogènes qui sont similaires mais pas identiques à ceux que nous avons rencontrés auparavant. De telles infections hétérologues ne sont pas l'objet de beaucoup d'études et certains exemples indiquent même qu'une immunité préexistante partielle peut mener à une aggravation de la maladie. Nous avons étudié le répertoire des lymphocytes T CD8 qui sont générés lors d'une rencontre avec un nouvel antigène, et ce en comparant infection primaire et secondaire. En utilisant le modèle expérimental d'infections à Listeria monocytogenes, nous avons pu montrer que lors d'une infection primaire, un répertoire diversifié comprenant des cellules T CD8 de forte et faible affinité est constitué. Au contraire, dans le cas d'une infection secondaire, le répertoire des cellules T est fortement limité et seulement les lymphocytes T de forte affinité sont impliqués dans la réponse immunitaire. Nous avons pu démontrer que ces Rangements sont provoqués par des cellules T CD8 mémoires capables de reconnaître un antigène présent dans les deux infections. Cette augmentation du seuil d'activation des cellules effectrices est majoritairement causée par les lymphocytes T CD8 mémoires non transférables. Ces observations indiquent que les vaccins visant à induire des cellules T anti-tumorales de faible affinité seraient inefficaces si le vaccin contient des épitopes contre lesquels il existe une mémoire immunologique. Les réponses immunitaires conduites par les cellules T contre les antigènes tumoraux dépendent des cellules T CD8 de faible réactivité contre les antigènes tumoraux puisque les cellules à forte réactivité sont éliminées par les mécanismes de tolérance. Nous basant sur l'existence dans la littérature de preuves indiquant que PTPN2 influence la réponse des cellules T de faible affinité, nous nous sommes intéressés à comprendre comment PTPN2 impacte les réponses des cellules T CD8 en général. Nous avons remarqué que des cellules T CD8 déficientes en PTPN2 exhibent une meilleure capacité à proliférer suite à une faible ou courte stimulation du récepteur des lymphocytes T. La phase effectrice est prolongée et la contraction retardée résultant ainsi à globalement plus de cellules effectrices. Ce phénomène est également accompagné d'une meilleure survie des cellules effectrices de différentiation terminale. Une fois transférées dans un nouvel hôte receveur, les cellules effectrices terminales KLRG1+CD127- déficientes en phosphatase PTPN2 peuvent survivre et se transformer en cellules mémoires CD127+ fonctionnelles. De façon inattendue, nous avons découvert que l'élimination de PTPN2 améliore l'efficacité du transfert et la formation des cellules mémoires ainsi que leur capacité protectrice. Manipuler l'activité de cette phosphatase apparaît donc comme une approche intéressante et prometteuse pour la thérapie cellulaire par transfert adoptif de lymphocytes T. Nos observations montrent que la manipulation d'un facteur intrinsèque, l'absence de PTPN2, peut, dans certaines circonstances, améliorer la réponse des cellules T. Une meilleure connaissance des mécanismes contrôlant la réponse des lymphocytes T CD8 pourrait donc permettre la manipulation de ces derniers et conduire à des réponses immunitaires plus vigoureuses. Si ces réponses sont déclenchées par l'utilisation de vaccins, il est nécessaire de considérer l'historique d'une exposition préalable à des agents pathogènes ou à des vaccins puisque celle-ci peut, comme nous l'avons démontré, influencer le répertoire des cellules T recrutées dans la réponse immunitaire et, par conséquent, modifier l'aptitude de notre système immunitaire à faire face à une infection. -- Our immune system plays an important role in the protection from disease. CD8 T cells are critical for the control of primary infections with most viruses and certain bacteria as well as against some tumors. Therefore, better knowledge of CD8 T cell responses might enable us to generate vaccines against pathogens for which currently no vaccines are available or to improve anti-tumor immune responses. In the first part of this thesis we addressed the issue how previously acquired immunity impacts on the response of CD8 T cells. We are often exposed to pathogens that are related but not identical to the previously encountered ones. Such heterologous infections are not well studied and there are some indications that partial pre-existing immunity may in some cases even lead to an enhancement of disease. We specifically studied the T cell repertoire of CD8 T cells that are responding to a newly encountered antigen in secondary compared to primary infections. Using the experimental model of Listeria monocytogenes infections, we showed that in primary infections a wide repertoire including high and low affinity CD8 T cells is recruited into the immune response. In contrast to this, in secondary infections, the T cell repertoire is severely restricted and only T cells of high affinity are responding. We were able to pinpoint this difference to the presence of memory CD8 T cells that recognize an antigen that is shared between the two subsequent infections. This increase in the activation threshold was most effectively mediated via non-transferable memory CD8 T cells. This would argue that vaccines targeting low affinity tumor-specific T cells would fail if the vaccine contains previously encountered CD8 T cell epitopes. T cell mediated immune responses to tumor antigen rely often on T cells which weakly react to tumor antigen as high affinity T cells are eliminated by tolerance mechanisms. Following indication in the literature that PTPN2 impacts on the response of such weakly antigen-reactive T cells, we investigated how PTPN2 impacts in general the response of CD8 T cells. We observed that CD8 T cells lacking PTPN2 show an enhanced expansion following weak or short-term T cell receptor stimulation. The effector phase is prolonged and contraction delayed thus resulting in overall more effector cells. This is accompanied by a better survival of terminal effector cells. When transferred into new recipients, KLRG1+CD127- terminal effector cells lacking PTPN2 can survive and convert into CD127+ functional memory cells. Surprisingly, we discovered that elimination of PTPN2 enhances the transfer efficacy and formation of memory cells as well as the protective capacity. Targeting PTPN2 might thus be a promising approach for adoptive T cell therapy. Our observations show how the manipulation of an intrinsic factor, the absence of PTPN2, can enhance T cell responses under certain circumstances. A better understanding of underlying mechanisms for the control of CDS T cell responses might enable the manipulation of these and allow for more powerful responses. If these responses are induced through vaccines it is imperative that the previous history of exposure to pathogens or vaccines is considered as it can, as we have shown in this thesis, influence the recruited T cell repertoire and thus possibly the ability to handle the infection.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

Immunogenicity of dimorphic and C-terminal fragments of Plasmodium falciparum MSP2 formulated with different adjuvants in mice.

BACKGROUND: Plasmodium falciparum MSP2 is a blood stage protein that is associated with protection against malaria. It was shown that the MSP2 dimorphic (D) and constant (C) regions were well recognized by immune human antibodies, and were characterized by major conserved epitopes in different endemic areas and age groups. These Abs recognized merozoite-derived proteins in WB and IFA. Here, the goal was to determine in mice the immunogenicity of the two allelic MSP2 D and C domains formulated with different adjuvants, for their possible use in future clinical studies. METHOD: Female A/J, C3H, and ICR mice were immunized subcutaneously 3 times at 3-week interval with a mixture of allelic and conserved MSP2 long synthetic peptides formulated with different adjuvants. One week after the third injection, sera from each group were obtained and stored at -20°C for subsequent testing. RESULTS: Both domains of the two MSP2 families are immunogenic and the fine specificity and intensity of the Ab responses are dependent on mouse strains and adjuvants. The major epitopes were restricted to the 20-mer peptide sequences comprising the last 8aa of D and first 12aa of C of the two allelic families and the first 20aa of the C region, this for most strains and adjuvants. Strong immune responses were associated with GLA-SE adjuvant and its combination with other TLR agonists (CpG or GDQ) compared to alhydrogel and Montanide. Further, the elicited Abs were also capable of recognizing Plasmodium-derived MSP2 and inhibiting parasite growth in ADCI. CONCLUSION: The data provide a valuable opportunity to evaluate in mice different adjuvant and antigen formulations of a candidate vaccine containing both MSP2 D and C fragments. The formulations with GLA-SE seem to be a promising option to be compared with the alhydrogel one in human clinical trials.

Characterization of local and systemic immune-modulation in melanoma patients

Le mélanome cutané est un des cancers les plus agressifs et dont l'incidence augmente le plus en Suisse. Une fois métastatique, le pronostic de survie moyenne avec les thérapies actuelles est d'environ huit mois, avec moins de 5% de survie à cinq ans. Les récents progrès effectués dans la compréhension de la biologie de la cellule tumorale mais surtout dans l'importance du système immunitaire dans le contrôle de ce cancer ont permis le développement de nouveaux traitements novateurs et prometteurs. Ces thérapies, appelées immunothérapies, reposent sur la stimulation et l'augmentation de la réponse immunitaire à la tumeur. Alors que les derniers essais cliniques ont démontré l'efficacité de ces traitements chez les patients avec des stades avancés de la maladie, le contrôle de la maladie à long- terme est seulement atteint chez une minorité des patients. La suppression locale et systémique de la réponse immunitaire spécifique anti-tumorale apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Des études sur les souris ont montré que les vaisseaux lymphatiques joueraient un rôle primordial dans ce processus en induisant une tolérance immune, ce qui permettrait à la tumeur d'échapper au contrôle du système immunitaire et métastatiser plus facilement. Ces excitantes découvertes n'ont pas encore été établi et prouvé chez l'homme. Dans cette thèse, nous montrons pour la première fois que les vaisseaux lymphatiques sont directement impliqués dans la modulation de la réponse immunitaire au niveau local et systémique dans le mélanome chez l'homme. Ces récentes découvertes montrent le potentiel de combiner des thérapies visant le système lymphatique avec les immunothérapies actuellement utilisées afin d'améliorer le pronostic des patients atteint du mélanome. -- Cutaneous melanoma is one of the most invasive and metastatic human cancers and causes 75% of skin cancer mortality. Current therapies such as surgery and chemotherapy fail to control metastatic disease, and relapse occurs frequently due to microscopic residual lesions. It is, thus, essential to develop and optimize novel therapeutic strategies to improve curative responses in these patients. In recent decades, tumor immunologists have revealed the development of spontaneous adaptive immune responses in melanoma patients, leading to the accumulation of highly differentiated tumor-specific T cells at the tumor site. This remains one of the most powerful prognostic markers to date. Immunotherapies that augment the natural function of these tumor-specific T cells have since emerged as highly attractive therapeutic approaches to eliminate melanoma cells. While recent clinical trials have demonstrated great progress in the treatment of advanced stage melanoma, long-term disease control is still only achieved in a minority of patients. Local and systemic immune suppression by the tumor appears to be responsible, in part, for this poor clinical evolution. These facts underscore the need for a better analysis and characterization of immune- related pathways within the tumor microenvironment (TME), as well as at the systemic level. The overall goal of this thesis is, thus, to obtain greater insight into the complexity and heterogeneity of the TME in human melanoma, as well as to investigate immune modulation beyond the TME, which ultimately influences the immune system throughout the whole body. To achieve this, we established two main objectives: to precisely characterize local and systemic immune modulation (i) in untreated melanoma patients and (ii) in patients undergoing peptide vaccination or checkpoint blockade therapy with anti-cytotoxic T- lymphocyte-asisctaed protein-4 (CTLA-4) antibody. In the first and main part of this thesis, we analyzed lymphatic vessels in relation to anti-tumor immune responses in tissues from vaccinated patients using a combination of immunohistochemistry (IHC) techniques, whole slide scanning/analysis, and an automatic quantification system. Strikingly, we found that increased lymphatic vessel density was associated with high expression of immune suppressive molecules, low functionality of tumor-infiltrating CD8+ T cells and decreased cytokine production by tumor-antigen specific CD8+ T cells in the blood. These data revealed a previously unappreciated local and systemic role of lymphangiogenesis in modulating T cell responses in human cancer and support the use of therapies that target lymphatic vessels combined with existing and future T cell based therapies. In the second objective, we describe a metastatic melanoma patient who developed pulmonary sarcoid-like granulomatosis following repetitive vaccination with peptides and CpG. We demonstrated that the onset of this pulmonary autoimmune adverse event was related to the development of a strong and long-lasting tumor-specific CD8+ T cell response. This constitutes the first demonstration that a new generation tumor vaccine can induce the development of autoimmune adverse events. In the third objective, we assessed the use of Fourier Transform Infrared (FTIR) imaging to identify melanoma cells and lymphocyte subpopulations in lymph node (LN) metastasis tissues, thanks to a fruitful collaboration with researchers in Brussels. We demonstrated that the different cell types in metastatic LNs have different infrared spectral features allowing automated identification of these cells. This technic is therefore capable of distinguishing known and novel biological features in human tissues and has, therefore, significant potential as a tool for histopathological diagnosis and biomarker assessment. Finally, in the fourth objective, we investigated the role of colony- stimulating factor-1 (CSF-1) in modulating the anti-tumor response in ipilimumab-treated patients using IHC and in vitro co-cultures, revealing that melanoma cells produce CSF-1 via CTL-derived cytokines when attacked by cytotoxic T lymphocytes (CTLs), resulting in the recruitment of immunosuppressive monocytes. These findings support the combined use of CSF-1R blockade with T cell based immunotherapy for melanoma patients. Taken together, our results reveal the existence of novel mechanisms of immune modulation and thus promote the optimization of combination immunotherapies against melanoma. -- Le mélanome cutané est un des cancers humains les plus invasifs et métastatiques et est responsable de 75% de la mortalité liée aux cancers de la peau. Les thérapies comme la chirurgie et la chimiothérapie ont échoué à contrôler le mélanome métastatique, par ailleurs les rechutes sous ces traitements ont été montrées fréquentes. Il est donc essentiel de développer et d'optimiser de nouvelles stratégies thérapeutiques pour améliorer les réponses thérapeutiques de ces patients. Durant les dernières décennies, les immunologistes spécialisés dans les tumeurs ont démontré qu'un patient atteint du mélanome pouvait développer spontanément une réponse immune adaptative à sa tumeur et que l'accumulation de cellules T spécifiques tumorales au sein même de la tumeur était un des plus puissants facteurs pronostiques. Les immunothérapies qui ont pour but d'augmenter les fonctions naturelles de ces cellules T spécifiques tumorales ont donc émergé comme des approches thérapeutiques très attractives pour éliminer les cellules du mélanome. Alors que les derniers essais cliniques ont démontré un progrès important dans le traitement des formes avancées du mélanome, le contrôle de la maladie à long-terme est seulement atteint chez une minorité des patients. La suppression immune locale et systémique apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Ces considérations soulignent la nécessité de mieux analyser et caractériser les voies immunitaires non seulement au niveau local dans le microenvironement tumoral mais aussi au niveau systémique dans le sang des patients. Le but de cette thèse est d'obtenir une plus grande connaissance de la complexité et de l'hétérogénéité du microenvironement tumoral dans les mélanomes mais aussi d'investiguer la modulation immunitaire au delà du microenvironement tumoral au niveau systémique. Afin d'atteindre ce but, nous avons établi deux objectifs principaux : caractériser précisément la modulation locale et systémique du système immunitaire (i) chez les patients atteints du mélanome qui n'ont pas reçu de traitement et (ii) chez les patients qui ont été traités soit par des vaccins soit par des thérapies qui bloquent les points de contrôles. Dans la première et majeure partie de cette thèse, nous avons analysé les vaisseaux lymphatiques en relation avec la réponse immunitaire anti-tumorale dans les tissus des patients vaccinés grâce à des techniques d'immunohistochimie et de quantification informatisé et automatique des marquages. Nous avons trouvé qu'une densité élevée de vaisseaux lymphatiques dans la tumeur était associée à une plus grande expression de molécules immunosuppressives ainsi qu'à une diminution de la fonctionnalité des cellules T spécifiques tumoral dans la tumeur et dans le sang des patients. Ces résultats révèlent un rôle jusqu'à là inconnu des vaisseaux lymphatiques dans la modulation directe du système immunitaire au niveau local et systémique dans les cancers de l'homme. Cette recherche apporte finalement des preuves du potentiel de combiner des thérapies visant le système lymphatique avec des autres immunothérapies déjà utilisées en clinique. Dans le second objectif, nous rapportons le cas d'un patient atteint d'un mélanome avec de multiples métastases qui a développé à la suite de plusieurs vaccinations répétées et consécutives avec des peptides et du CpG, un évènement indésirable sous la forme d'une granulomatose pulmonaire sarcoid-like. Nous avons démontré que l'apparition de cet évènement était intimement liée au développement d'une réponse immunitaire durable et spécifique contre les antigènes de la tumeur. Par là- même, nous prouvons pour la première fois que la nouvelle génération de vaccins est aussi capable d'induire des effets indésirables auto-immuns. Pour le troisième objectif, nous avons voulu savoir si l'utilisation de la spectroscopie infrarouge à transformée de Fourier (IRTF) était capable d'identifier les cellules du mélanome ainsi que les différents sous-types cellulaires dans les ganglions métastatiques. Grâce à nos collaborateurs de Bruxelles, nous avons pu établir que les diverses composantes cellulaires des ganglions atteints par des métastases du mélanome présentaient des spectres infrarouges différents et qu'elles pouvaient être identifiées d'une façon automatique. Cette nouvelle technique permettrait donc de distinguer des caractéristiques biologiques connues ou nouvelles dans les tissus humains qui auraient des retombées pratiques importantes dans le diagnostic histopathologique et dans l'évaluation des biomarqueurs. Finalement dans le dernier objectif, nous avons investigué le rôle du facteur de stimulation des colonies (CSF-1) dans la modulation de la réponse immunitaire anti-tumorale chez les patients qui ont été traités par l'Ipilimumab. Nos expériences in vivo au niveau des tissus tumoraux et nos co-cultures in vitro nous ont permis de démontrer que les cytokines secrétées par les cellules T spécifiques anti-tumorales induisaient la sécrétion de CSF-1 dans les cellules du mélanome ce qui résultait en un recrutement de monocytes immunosuppresseurs. Dans son ensemble, cette thèse révèle donc l'existence de nouveaux mécanismes de modulation de la réponse immunitaire anti-tumorale et propose de nouvelles optimisations de combinaison d'immunothérapies contre le mélanome.