Systems vaccinology : molecular signatures of immunity to Bordetella pertussis

The worldwide resurgence of whooping cough (pertussis), even in highly vaccinated populations, demands improved pertussis vaccines. In this thesis a systems vaccinology approach is applied to deepen knowledge of the immune responses evoked by different pertussis vaccines and compare this with a Bordetella pertussis infection since the latter induces robust protection. Infection-induced responses in mice conferred sterilizing protection that is caused by systemic immunity but more importantly by mucosal IgA, T-helper (Th)1/Th17 responses, and ‘trained’ innate immune cells in the lungs. An experimental outer membrane vesicle vaccine (omvPV) was compared with the two licensed vaccines, acellular vaccine (aPV), whole-cell vaccine (wPV) as well as a B. pertussis infection. OmvPV evoked a different immunoproteomic profile with respect to antibody levels, antigen specificity, and subclass distribution. Furthermore, omvPV confers equal protection in mice as wPV, but with a lower inflammatory response. In this thesis it is also shown that the immunization route is critical. Although subcutaneous omvPV immunization is effective, pulmonary administration lead to superior protection, comparable to infection-induced immunity and included hallmarks of protection such as pulmonary Th17 cells and mucosal IgA. The molecular and cellular signatures described in this thesis may have an important contribution to enhanced pertussis immunity

Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine

Drug Delivery Technolog

Decoding the complexities of human malaria through systems immunology

The complexity of the Plasmodium parasite and its life cycle poses a challenge to our understanding of the host immune response against malaria. Studying human immune responses during natural and experimental Plasmodium infections can enhance our understanding of malaria-protective immunity and inform the design of disease-modifying adjunctive therapies and next-generation malaria vaccines. Systems immunology can complement conventional approaches to facilitate our understanding of the complex immune response to the highly dynamic malaria parasite. In this review, recent studies that used systems-based approaches to evaluate human immune responses during natural and experimental Plasmodium falciparum and Plasmodium vivax infections as well as during immunization with candidate malaria vaccines are summarized and related to each other. The potential for next-generation technologies to address the current limitations of systems-based studies of human malaria are discussed

Caracterización de la respuesta inmunitaria inducida por nuevas vacunas contra la tuberculosis basadas en nanopartículas

Lung diseases are a common and diverse group of illnesses that represent a health problem affecting millions of people worldwide. The microenvironment and the immune system in the lung are very important but they are also the most challenging immunological dilemma for the host. On one hand, lung location and function are constantly exposed to pathogens; but on the other hand should be sterile and preserve lung homeostasis through the balance between tolerance and inflammation. For this reason is extremely important to characterize and understand the mechanisms and populations involved in a protective immune response, and find better targets to design vaccines. The aim of this project is to define and understand the immunological profiles that correlate with greater protection generated by preventive and therapeutic vaccines in the lung. Preventive vaccines are generally administered to healthy individuals to prevent or ameliorate infectious diseases, but therapeutic cancer vaccines are administrated once the individual has already developed the disease to strength patient's own immune responses. We will focus on two different lung diseases: an infection caused by a bacteria and lung cancer. To study a bacterial infection, we will use as a model Mycobacterium tuberculosis (Mtb), responsible for Tuberculosis (TB). TB is a serious global health problem and the only licensed vaccine against TB is "Mycobacterium bovis Bacille Calmette-Guérin" (BCG). But BCG has a poor efficacy. To achieve a better understanding of the type of immune responses that confer protection, we will use a mouse model to assess the changes in the immune system produced in the lung by vaccines and in response to Mtb infection. Lung cancer is a devastating disease and a major therapeutic burden with poor survival rates. Cancer cells have been in the focus of the research, but the importance of the immune system is becoming more important. Syngenic murine models entailing the injection of immunological compatible cancer cells in immunocompetent mice will allow us to study of the modulation of the immune system performed by cancer cells. Moreover, we will use therapeutic vaccines against tumor cells to activate anti-tumor T cells to recognize and destroy the tumor, increasing the effectiveness when used alone or in combination with conventional therapies. In both approaches, the immune cell populations present in the bronchoalverolar lavage (BAL) and in the lung parenchyma, and the ones induced by the different experimental conditions, will be analyzed and characterized by flow cytometry and ELISA. Also, cells will be obtained to perform proteomic and gene expression profiles to better define the pathways with impact in controlling infection or cancer cells. The outputs generated by our research would lead in the future to a more rational design of protective prophylactic and therapeutic vaccines against lung diseases.Las enfermedades pulmonares son un grupo diverso de enfermedades que afectan a millones de personas en todo el mundo. El microambiente y el sistema inmunitario en el pulmón son muy importantes a la vez que suponen un dilema para el huésped. Por un lado, por su ubicación y función, el pulmón está constantemente expuesto a patógenos; por otro lado debe ser estéril y preservar la homeostasis a través del equilibrio entre tolerancia e inflamación. Por ello es extremadamente importante caracterizar y comprender los mecanismos y las poblaciones involucradas en una respuesta inmunitaria protectora, y encontrar mejores dianas para diseñar vacunas. El objetivo de este proyecto es definir y comprender los perfiles inmunitarios que correlacionan con una mayor protección generada por vacunas preventivas y terapéuticas en pulmón. Las vacunas preventivas se administran a individuos sanos para prevenir o mejorar enfermedades infecciosas, mientras que las vacunas terapéuticas contra el cáncer se administran, para fortalecer la propia respuesta inmunitaria del paciente. Nos centraremos en dos enfermedades pulmonares: una infección causada por una bacteria y cáncer de pulmón. Para estudiar la infección bacteriana, utilizaremos como modelo Mycobacterium tuberculosis (Mtb), responsable de la tuberculosis. La tuberculosis es un grave problema de salud mundial. La única vacuna autorizada contra ella es "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que tiene una baja eficacia. Para comprender mejor el tipo de respuestas inmunológicas que confieren protección, utilizaremos un modelo de ratón para evaluar los cambios en el sistema inmunitario producidos por diferentes vacunas y la infección de Mtb en el pulmón. El cáncer de pulmón es una enfermedad devastadora, con una carga terapéutica importante y baja tasa de supervivencia. La investigación se ha centrado en las células tumorales, pero el sistema inmunitario se está volviendo cada vez más importante. Los modelos murinos sinogénicos permiten la inyección de células cancerosas inmunológicamente compatibles en ratones inmunocompetentes para estudiar la modulación del sistema inmunitario por las células tumorales. Además, utilizaremos vacunas terapéuticas para activar las células T a fin de que reconozcan y destruyan el tumor, aumentando su eficacia solas o en combinación con terapias convencionales. En ambos casos, las poblaciones celulares inmunitarias presentes en el lavado broncoalverolar y el parénquima pulmonar en las diferentes condiciones experimentales serán analizadas y caracterizadas por citometría de flujo y ELISA. Se obtendrán además células para estudios proteómicos y de expresión génica, para definir mejor las vías implicadas en el control de la infección o de las células cancerosas. Los resultados obtenidos podrían conducir en el futuro a un diseño más racional de vacunas profilácticas y terapéuticas contra enfermedades pulmonares.As enfermidades pulmonares son un grupo diverso de enfermidades que afectan a millóns de persoas en todo o mundo. O microambiente e o sistema inmunitario nos pulmóns son moi importantes ao mesmo tempo que representan un dilema para o hospedeiro. Por unha banda, debido á súa localización e función, o pulmón está constantemente exposto a patóxenos; por outra banda, debe ser estéril e preservar a homeostase a través do equilibrio entre tolerancia e inflamación. Por iso, é moi importante caracterizar e comprender os mecanismos e poboacións implicados nunha resposta inmune protectora e atopar mellores obxectivos para o deseño de vacinas. O obxectivo deste proxecto é definir e comprender os perfís inmunolóxicos que se correlacionan con maior protección xerada por vacinas preventivas e terapéuticas no pulmón. As vacinas preventivas son xeralmente administradas a individuos sans para previr ou mellorar doenzas infecciosas, mentres que as vacinas terapéuticas contra o cancro son administradas unha vez o individuo desenvolve a enfermidade para fortalecer a súa resposta inmunitaria. Centrarémonos en dúas enfermidades pulmonares: unha infección bacteriana e cancro de pulmón. Para estudar unha infección bacteriana, usaremos como modelo Mycobacterium tuberculosis (MTB), responsable da tuberculose (TB), un grave problema de saúde global. A única vacina con licenza actualmente é "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que ten unha baixa eficacia. Para entender mellor o tipo de respostas inmunolóxicas que confiran protección, usaremos un modelo de rato para avaliar cambios no sistema inmunitario causadas por vacinas e infección por MTB no pulmón. O cancro de pulmón é unha enfermidade devastadora, cunha importante carga terapéutica e baixa taxa de supervivencia. As células tumorais foron o foco da investigación, pero o sistema inmunolóxico é cada vez máis importante. Os modelos murinos sinxénicos, que inclúen a inxección de células de cancro imunolóxicamente compatibles en ratos inmunocompetentes, permítennos estudar a modulación do sistema inmunitario por células tumorais. Ademáis, utilizaremos vacinas terapéuticas para activar as células T que recoñezan e destrúan o tumor, aumentando a súa eficacia cando se usa só ou en combinación con terapias convencionais. En ambos casos, as poboacións de células inmunitarias presentes no lavado broncoalverolar e o parénquima pulmonar nas diferentes condicións experimentais analizaranse e caracterízanse por citometría de fluxo e ELISA. As células tamén se obterán para estudos proteómicos e de expresión xénica, para definir mellor as vías implicadas no control da infección ou as células cancerosas. Os resultados obtidos poderían levar no futuro a un deseño máis racional de vacinas profilácticas e terapéuticas contra as enfermidades pulmonares.Xunta de Galicia | Ref. ED431C 2016/041Xunta de Galicia and the European Regional Development Fund (ERDF) | Ref. ED431G2019/06Fundação para a Ciência e a Tecnologia | Ref. PTDC/SAU-INF/28463/2017Fundação para a Ciência e a Tecnologia | Ref. UIDB/50026/2020Fundação para a Ciência e a Tecnologia | Ref. UIDP/50026/2020FEDER | Ref. NORTE-01-0145-FEDER-000013FEDER | Ref. NORTE-01-0145- FEDER-00002

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.Drug Delivery Technolog

Sex Differences in Vaccine-Specific and Heterologous Immunity Following Administration of Measles and /or DtwP Vaccines to Nine Month-Old Gambian Infants

The expanded programme of immunization was introduced in The Gambia over 30 years ago, using empirically developed vaccines with limited understanding of exactly how they work. Observational studies showing vaccines have heterologous beneficial or deleterious effects on disease susceptibility, dependent on sex, have been controversial, with no immunological data to support these epidemiological findings. The live measles vaccine (MV) seems to have beneficial protective effects, while diphtheria, tetanus, whole cell pertussis (DTwP) vaccine increases disease susceptibility, both effects being more prominent in females. These effects are modified when the vaccines are given simultaneously. This thesis describes the results of a randomized trial primarily aimed at studying the heterologous effects of MV and DTwP vaccination of 9 month old Gambian infants. Assays included multiplex cytokine analysis of culture supernatants, flow cytometric intracellular staining, vaccine antibody assays, and whole transcriptome microarray analysis. Measles and DTwP antibodies were not affected by combining vaccines or by gender. When DTwP is given with MV, the measles-specific response was skewed away from a protective CD8 IFN-γ response, while TT responses become more pro-inflammatory suggesting that MV has an adjuvant effect on DTwP immunity. A striking finding was the immunosuppressive effect of DTwP, particularly in females. There was a reduction in pro-inflammatory cytokines and reduced IFN-γ:IL-10 ratios in aCD3/28 cultures, alongside down-regulated interferon response gene pathways, and increased innate IL-10 production in DTwP vaccinated females. By contrast, DTwP vaccinated males a more pro-inflammatory profile than females with only upregulated genes post-vaccination. Males vaccinated with MV+DTwP were less pro-inflammatory than females, and had only down-regulated genes suggesting this combination had opposite effects in males and females. The results provide plausible immunological explanations for the observed beneficial and deleterious effects of MV and DTwP respectively. These will now need to be tested in carefully designed prospective studies

Global mRNA and miRNA transcriptome profiling of peripheral blood mononuclear cells to investigate the host immunogenetic response to PRRSV vaccination in pigs

This dissertation aims to identify the candidate genes of the functional network of host immune response to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs; to explore the breed differences on vaccine induced transcriptional response between German Landrace (DL) and Pietrain (Pi) pigs; and to elucidate the post transcriptional regulatory mechanism of vaccine induced gene expression in the peripheral blood mononuclear cells (PBMCs). The Affymetrix gene chip microarray technique was employed for global expression profiling of messenger RNA (mRNA) and microRNA (miRNA) in PBMCs collected in a time series manner following PRRSV vaccination in purebred DL and Pi pigs. Additionally, microarray expression results were validated by qRT-PCR and the PRRSV-specific plasma antibody titre was monitored by ELISA. The PRRSV-specific plasma antibody titre indicated the piglets free from maternal antibody at the time of primary vaccination and rose above the threshold following two weeks of the primary vaccination that subsequently reached a plateau at four weeks post vaccination. The global mRNA profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated Landrace pigs at immediately before (0 h), and at 6, 24 and 72 h after PRRSV vaccination revealed a distinct host innate immune transcriptional response. A total of 14,231 transcripts were found to be expressed in PBMCs of vaccinated and unvaccinated pigs. Differential expression analysis (FDR ±1.5) identified 542, 2,263 and 357 differentially expressed genes at 6, 24 and 72 h post vaccination. APP, TRAF6, PIN1, FOS, CDKN1A and TNFAIP3 identified to be potential candidate genes for early stage PRRSV vaccine response in Landrace pigs. In Pietrain pigs, 295 and 116 transcripts were found to be differentially expressed in PBMCs at 1 and 28 days post vaccination, respectively. This study suggested that the innate immune transcriptional network is likely to be regulated by LCK, STAT3, ATP5B, UBB and RSP17; while TGFβ1, IL7R, RAD21, SP1 and GZMB were found to be predictive for the adaptive immune transcriptional response to PRRSV vaccine in PBMCs of Pi pigs. The global microRNA profiles of PBMCs identified 12, 259 and 14 differentially expressed (DE) miRNAs in DL; and 0, 222 and 13 DE miRNAs in Pietrain at 6, 24 and 72 h post vaccination, respectively. There were remarkable differences on expression dynamics of both mRNAs and miRNAs between DL and Pi pigs. Integrated mRNA-miRNA network revealed the inverse correlation between vaccine induced altered mRNAs and miRNAs in PBMCs. Results of this immunogenomics study advances our understanding on the genetic control of PRRS.Erstellung von globalen mRNA und miRNA Transkriptomprofilen in mononukleäre Zellen des peripheren Blutes zur Untersuchung der Wirts immunogenetischen Reaktion auf eine PRRSV Impfung bei Schweinen Die vorliegende Arbeit zielt darauf ab, Kandidatengene des funktionellen Netzwerks der wirtsspezifischen Immunantwort auf den PRRS-Virus (PRRSV) Impfstoff bei Schweinen zu identifizieren; um transkriptionale Unterschiede durch den induzierten Impfstoff in den zwei Schweinerassen Deutschen Landrasse (DL) und Piétrain (Pi) zu erkunden; und die Aufklärung von Post-transkriptionellen Mechanismen bedingt durch den Impfstoff in den mononukleären Zellen des peripheren Blutes (PBMCs). Zur Erstellung der Transkriptomprofile der Boten-RNA (mRNA) sowie der microRNA (miRNA) in reinrassigen DL und Pi Schweinen zu unterschiedlichen Zeitpunkten nach der PRRSV Impfung wurde die Affymetrix Gen-Chip-Microarray-Technik eingesetzt. Zusätzlich wurden die Microarray Ergebnisse mittels qRT-PCR validiert und die PRRSV-spezifischen Plasma Antikörpertiter durch ELISA bestimmt. Durch den PRRSV-spezifischen Plasma Antikörpertiter zeigte sich, dass die Ferkel frei von mütterlichen Antikörpern zum Zeitpunkt der Erstimpfung waren. Nach der ersten Impfung stieg der Titer in den folgenden zwei Wochen über dem Grenzwert, und erreichte sein Plateau vier Wochen nach der Impfung. Die Betrachtung der globalen mRNA Profile von PBMCs von PRRSV geimpft und ungeimpften DL Schweinen unmittelbar vor 0 und mit 6, 24 und 72 h nach der Impfung ergab eine deutlich angeborene transkriptionelle Wirts Immunreaktion. Insgesamt waren 14.231 Transkripte in PBMCs von geimpften und nicht geimpften Schweine exprimiert. Die Expressionsanalyse (FDR ± 1,5) identifiziert 542, 2263 und 357 differentiell exprimierte Gene 6, 24 und 72 h nach der Impfung. Als potenzielle Kandidatengene für das frühe Stadium der Impfreaktion konnten APP, TRAF6, PIN1, FOS, CDKN1A und TNFAIP3 identifiziert werden. In Piétrain Schweinen waren 295 und 116 Transkripte in PBMCs an Tag 1 und 28 nach der Impfung unterschiedlich exprimiert. Diese Ergebnisse zeigen, dass das angeborene Immunnetzwerk wahrscheinlich durch LCK, STAT3, ATP5B, UBB und RSP17 geregelt wird; während sich TGFβ1, IL7R, Rad21, SP1 und GZMB für die adaptive Immunreaktion auf den PRRSV-Impfstoff in PBMCs von Pi-Schweinen als prädiktiv erwiesen. Die microRNA-Profile von PBMCs identifiziert 12, 259 und 14 unterschiedlich exprimiert miRNAs in DL; und 0, 222 und 13 miRNAs in Pi, 6, 24 und 72 h nach der Impfung. Es gab deutliche Unterschiede bei der Expressionsdynamik sowohl bei der mRNAs als auch miRNAs zwischen DL und Pi Schweine. Integrierte mRNA-miRNA-Netzwerke zeigen eine inverse Korrelation zwischen der durch den Impfstoff induzierten veränderten mRNAs und miRNAs Expression in PBMCs. Die Ergebnisse dieser immunogenomischen Studie erweitert unser Verständnis über die genetische Kontrolle von PRRS

Inflammation, Immunity, and Vaccines for Helicobacter

Helicobacter pylori represents the major etiologic agent of gastritis, gastric, and duodenal ulcer disease and can cause gastric cancer and mucosa-associated lymphoid tissue B-cell lymphoma. It is clear that the consequences of infection reflect diverse outcomes of the interaction of bacteria and host immune system. The hope is that by deciphering the deterministic rules – if any – of this interplay, we will eventually be able to predict, treat, and ultimately prevent disease. Over the past year, research on the immunology of this infection started to probe the role of small noncoding RNAs, a novel class of immune response regulators. Furthermore, we learned new details on how infection is detected by innate pattern recognition receptors. Induction of effective cell-mediated immunity will be key for the development of a vaccine, and new work published analyzed the relevance and contribution of CD4 T helper cell subsets to the immune reaction. Th17 cells, which are also induced during natural infection, were shown to be particularly important for vaccination. Cost-efficiency of vaccination was re-assessed and confirmed. Thus, induction and shaping of the effector roles of such protective Th populations will be a target of the newly described vaccine antigens, formulations, and modes of application that we also review here

Interferon-driven alterations of the host’s amino acid metabolism in the pathogenesis of typhoid fever

Enteric fever, caused by Salmonella enterica serovar Typhi, is an important public health problem in resource-limited settings and, despite decades of research, human responses to the infection are poorly understood. In 41 healthy adults experimentally infected with wild-type S. Typhi, we detected significant cytokine responses within 12 h of bacterial ingestion. These early responses did not correlate with subsequent clinical disease outcomes and likely indicate initial host–pathogen interactions in the gut mucosa. In participants developing enteric fever after oral infection, marked transcriptional and cytokine responses during acute disease reflected dominant type I/II interferon signatures, which were significantly associated with bacteremia. Using a murine and macrophage infection model, we validated the pivotal role of this response in the expression of proteins of the host tryptophan metabolism during Salmonella infection. Corresponding alterations in tryptophan catabolites with immunomodulatory properties in serum of participants with typhoid fever confirmed the activity of this pathway, and implicate a central role of host tryptophan metabolism in the pathogenesis of typhoid fever