Chagas Disease Diagnostic Applications: Present Knowledge and Future Steps

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong and debilitating illness of major significance throughout Latin America and an emergent threat to global public health. Being a neglected disease, the vast majority of Chagasic patients have limited access to proper diagnosis and treatment, and there is only a marginal investment into R&D for drug and vaccine development. In this context, identification of novel biomarkers able to transcend the current limits of diagnostic methods surfaces as a main priority in Chagas disease applied research. The expectation is that these novel biomarkers will provide reliable, reproducible and accurate results irrespective of the genetic background, infecting parasite strain, stage of disease, and clinical-associated features of Chagasic populations. In addition, they should be able to address other still unmet diagnostic needs, including early detection of congenital T. cruzi transmission, rapid assessment of treatment efficiency or failure, indication/prediction of disease progression and direct parasite typification in clinical samples. The lack of access of poor and neglected populations to essential diagnostics also stresses the necessity of developing new methods operational in point-of-care settings. In summary, emergent diagnostic tests integrating these novel and tailored tools should provide a significant impact on the effectiveness of current intervention schemes and on the clinical management of Chagasic patients. In this chapter, we discuss the present knowledge and possible future steps in Chagas disease diagnostic applications, as well as the opportunity provided by recent advances in high-throughput methods for biomarker discovery.Fil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Fernandez Aguero, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Comparative systemic analysis of human immunoglobulin repertoires

The humoral immune system is majorly composed of B cells producing effector immunoglobulin molecules, the vast diversity of which allow for the neutralization of pathogenic threats never previously seen by the immune system. High-throughput sequencing technology has allowed this vast repertoire to be characterized and quantified, but understanding this complex system requires methods of comparison to identify and differentiate B cell populations. In this thesis, differences between groups of repertoires within individuals are analyzed at both the cellular and proteomic level. Novel experimental techniques and visualization methods will allow for the analyses of several such high-dimensional complex systems, leading to a fuller picture of the B cells’ contribution to the immune system.Microbiolog

Array based genetic profiling of chronic lymphocytic leukemia

Although no common genetic defect has been described in chronic lymphocytic leukemia (CLL), recurrent genomic aberrations (i.e. deletions of chromosome 11q, 13q, 17p and trisomy 12) are important for prognostication. Deletion of 13q as single aberration is associated with the best prognosis, whereas del(11q) and del(17p) predict a poor outcome. Recent development of high-resolution genomic techniques, i.e. microarrays, provides effective detection of the known recurrent aberrations and simultaneous exploration of the whole genome. Hence, this thesis aimed to map genomic aberrations in CLL through application of high-resolution microarrays. In paper I, we investigated the pros and cons of four differently designed microarray platforms. All platforms readily detected the known recurrent aberrations in CLL as well as other large copy-number aberrations (CNAs). The difference in technical performance and discrepancies in detection of small CNAs were influenced by differential platform density, different reference sets, and platform-specific analysis. In paper II and IV, 250K single nucleotide polymorphism (SNP)-array screening of 203 and 370 samples, respectively, detected CNAs in >90% and the known recurrent aberrations in >70% of patients. Moreover, novel recurrent gains of chromosome 2p in combination with 11q deletion and copy-number neutral loss of heterozygosity on chromosome 13q were revealed. Furthermore, a high genomic complexity was correlated to worse survival, but also closely linked to poor-prognostic markers. In addition, study IV also included follow-up samples (n=43) to investigate clonal evolution, which showed that patients with unmutated immunoglobulin heavy chain variable (IGHV) genes and treated patients with mutated IGHV genes often gained novel aberrations. In paper III, high-density screening revealed a different spectrum of genomic aberrations in CLL patients with ‘stereotyped’ IGHV3-21 (poor-prognostic) versus IGHV4-34 (good-prognostic) B-cell receptors. IGHV3-21 subset #2 (n=29) showed a high frequency of samples carrying genomic aberrations, and a particularly high prevalence of del(13q) and del(11q), which may correspond to the adverse survival reported for these patients. In contrast, IGHV4-34 subset #4 (n=17) showed a lower frequency and complexity of CNAs, which may reflect an inherent low-proliferative disease, preventing accumulation of genomic alterations. In summary, the studies included in this thesis provided a greater insight of genomic aberrations in newly diagnosed patients, at follow-up and in different subgroups of CLL

Proteomics in biomarker discovery and drug development

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub-proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time can be considered in an integrated way. Proteomic technology has been extensively used to tackle a wide variety of medical subjects including biomarker discovery and drug development. By complement with other new technique advances in genomics and bioinformatics, proteomics has a great potential to make considerable contribution to biomarker identification and to revolutionize drug development process. This article provides a brief overview of the proteomic technologies and their application in biomarker discovery and drug development.postprin

지도 학습 기반 바이오패닝 클론 증폭 패턴 분석을 통한 항원 결합 반응성 예측

학위논문(박사) -- 서울대학교대학원 : 의과대학 의과학과, 2021.8. 정준호.Background: Monoclonal antibodies (mAbs) are produced by B cells and specifically binds to target antigens. Technical advances in molecular and cellular cloning made it possible to purify recombinant mAbs in a large scale, enhancing the multiple research area and potential for their clinical application. Since the importance of therapeutic mAbs is increasing, mAbs have become the predominant drug classes for various diseases over the past decades. During that time, immense technological advances have made the discovery and development of mAb therapeutics more efficient. Owing to advances in high-throughput methodology in genomic sequencing, phenotype screening, and computational data analysis, it is conceivable to generate the panel of antibodies with annotated characteristics without experiments. Thesis objective: This thesis aims to develop the next-generation antibody discovery methods utilizing high-throughput antibody repertoire sequencing and bioinformatics analysis. I developed novel methods for construction of in vitro display antibody library, and machine learning based antibody discovery. In chapter 3, I described a new method for generating immunoglobulin (Ig) gene repertoire, which minimizes the amplification bias originated from a large number of primers targeting diverse Ig germline genes. Universal primer-based amplification method was employed in generating Ig gene repertoire then validated by high-throughput antibody repertoire sequencing, in the aspect of clonal diversity and immune repertoire reproducibility. A result of this research work is published in ‘Journal of Immunological Methods (2021). doi: 10.1016/j.jim.2021. 113089’. In chapter 4, I described a novel machine learning based antibody discovery method. In conventional colony screening approach, it is impossible to identify antigen specific binders having low clonal abundance, or hindered by non-specific phage particles having antigen reactivity on p8 coat protein. To overcome the limitations, I applied the supervised learning algorithm on high-throughput sequencing data annotated with binding property and clonal frequency through bio-panning. NGS analysis was performed to generate large number of antibody sequences annotated with its’ clonal frequency at each selection round of the bio-panning. By using random forest (RF) algorithm, antigen reactive binders were predicted and validated with in vitro screening experiment. A result of this research work is published in ‘Experimental & Molecular Medicine (2017). doi:0.1038/emm.2017.22’ and ‘Biomolecule (2020). doi:10.3390/biom10030421’. Conclusion: By combining conventional antibody discovery techniques and high-throughput antibody repertoire sequencing, it was able to make advances in multiple attributes of the previous methodology. Multi-cycle amplification with Ig germline gene specific primers showed the high level of repertoire distortion, but could be improved by employing universal primer-based amplification method. RF model generates the large number of antigen reactive antibody sequences having various clonal enrichment pattern. This result offers the new insight in interpreting clonal enrichment process, frequency of antigen specific binder does not increase gradually but depends on the multiple selection rounds. Supervised learning-based method also provides the more diverse antigen specific clonotypes than conventional antibody discovery methods.연구의 배경: 단일 클론 항체 (monoclonal antibody, mAb) 는 B 세포에서 생산되어 표적 항원에 특이적으로 결합하는 폴리펩타이드 복합체 이다. 분자 및 세포 클로닝 기술의 발전으로 재조합 단일 클론 항체를 대용량으로 생산하는것이 가능해졌으며, 이를 바탕으로 다양한 연구 및 임상 분야에서의 활용이 확대되고 있다. 또한 치료용 항체를 효율적으로 발굴하고 개발하는 기술에 대한 비약적인 발전이 이루어졌다. 유전자 서열 분석, 표현형 스크리닝, 컴퓨팅 기반 분석법 분야에서 이루어진 고집적 방법론 (high-throughput methodology) 의 발전과 이의 응용을 통해, 비실험적 방법을 통해 항원 반응성 항체 패널을 생산하는것이 가능해졌다. 연구의 목표: 본 박사 학위 논문은 고집적 항체 레퍼토어 시퀀싱 (high-throughput antibody repertoire sequencing) 과 생물정보학 (bioinformatics) 기법을 활용하여 신규한 (novel) 차세대 항체 발굴법 (next-generation antibody discovery method) 을 개발하는것을 목표로 하고 있다. 본 연구를 통해 in vitro display 항체 라이브러리를 제작하기 위한 신규 프로토콜 및 기계 학습을 기반으로한 항체 발굴법을 개발 하였다. Chapter 3: 항체 레퍼토어를 증폭하는 과정에서, 다수의 생식세포 면역 글로불린 유전자 (germline immunoglobulin gene) 특이적 프라이머 사용에 의해 발생하는 증폭 편차 (amplification bias) 를 최소화 하는 방법론에 대해 기술하였다. 유니버셜 (universal) 프라이머를 사용한 다중 사이클 증폭 (multi-cycle amplification) 법이 사용되었으며, 고집적 항체 레퍼토어 시퀀싱을 통해, 클론 다양성 (clonal diversity) 및 면역 레퍼토어 재구성도 (immune repertoire reproducibility) 를 생물정보학적 기법으로 측정하여 신규 방법론에 대한 검증을 수행하였다. 본 연구의 연구결과는 다음의 학술지에 출판 되었다: Journal of Immunological Methods (2021). doi: 10.1016/j.jim.2021. 113089. Chapter 4: 기계 학습 기반의 항체 발굴법 개발에 대해 기술하였다. 전통적 콜로니 스크리닝 (colony screening) 방법에서는, 클론 빈도 (clonal abundance) 가 낮은 클론을 발굴 하거나 선택압 (selective pressure) 이 부여되는 과정에서, p8 표면 단백질의 비 특이적 항원 특이성을 제거할 수 없다. 이러한 제한점을 극복하기 위해서 항원 결합능 및 바이오패닝 에서의 클론 빈도가 측정 되어있는 고집적 항체 서열 데이터를 대상으로 지도 학습 알고리즘을 적용하였다. 랜덤 포레스트 (random forest, RF) 알고리즘을 적용하여 항원 특이적 항체 클론을 예측하였으며, 시험관 내 스크리닝을 통해 항원 특이성을 검증하였다. 본 연구의 연구 결과는 다음의 학술지에 출판되었다: 1) Experimental & Molecular Medicine (2017). doi:0.1038/emm.2017.22., 2) Biomolecule (2020). doi:10.3390/biom10030421. 결론: 전통적 항체 발굴 기술과 고집적 항체 레퍼토어 시퀀싱 기술을 융합함으로써, 기존 방법론의 다양한 한계점을 개선할 수 있었다. 면역 글로불린 생식세포 유전자 특이적 프라이머를 사용한 다중 사이클 증폭은 클론 빈도 및 다양성에 왜곡을 유도 하였으나, 유니버셜 프라이머를 사용한 증폭법을 통해 높은 효율로 레퍼토어 왜곡을 개선시킬 수 있음을 관찰할 수 있었다. RF 모델은 다양한 클론 증폭 패턴 (enrichment pattern) 을 가지는 항원 반응성 항체 서열을 생성하였다. 이를 통해 항원에 특이적으로 결합하는 클론이 단계적으로 증폭되는 것이 아니라 초기 및 후기의 다수의 선별 단계 (selection round) 에 의존함을 확인할 수 있었으며, 바이오패닝 에서의 클론 증폭에 대한 새로운 해석을 제시하였다. 또한 지도 학습을 기반으로 발굴 된 클론들에서, 전통적 콜로니 스크리닝 방법과 대비하여 더 높은 서열 다양성을 관찰할 수 있었다.1. Introduction 8 1.1. Antibody and immunoglobulin repertoire 8 1.2. Antibody therapeutics 16 1.3. Methodology: antibody discovery and engineering 21 2. Thesis objective 28 3. Establishment of minimally biased phage display library construction method for antibody discovery 29 3.1. Abstract 29 3.2. Introduction 30 3.3. Results 32 3.4. Discussion 44 3.5. Methods 47 4. In silico identification of target specific antibodies by high-throughput antibody repertoire sequencing and machine learning 58 4.1. Abstract 58 4.2. Introduction 60 4.3. Results 64 4.4. Discussion 111 4.5. Methods 116 5. Future perspectives 129 6. References 135 7. Abstract in Korean 150박

Epstein-Barr virus: environmental trigger and therapeutic target in autoimmune diseases

Das Epstein-Barr-Virus (EBV) ist ein ubiquitäres Virus, mit welchem über 95% aller Menschen infiziert sind. Die meisten Träger des Pathogens bleiben während ihrer frühen Kindheit asymptomatisch. Eine Minderheit derer, die zu einem späteren Zeitpunkt infiziert werden, durchlaufen als symptomatische Primärinfektion die infektiöse Mononukleose (IM). Nach einer lytischen Replikation während der Primärinfektion bleibt EBV latent und lebenslang in B-Zellen des betroffenen Organismus. Die Mehrheit der EBV-Träger ist durch das Virus nicht beeinträchtigt. Allerdings ist für einen Teil der EBV-Infektionen und im besonderen der IM eine Assoziation mit einer Vielzahl von Krankheiten beschrieben. Zu diesen Erkrankungen zählen das Burkitt- und Hodgkin-Lymphom, proliferative Krankheiten nach Transplantationen und das Nasopharynxkarzinom. Zusätzlich liegen zahlreiche Hinweise für einen Zusammenhang von EBV mit verschiedenen Autoimmunkrankheiten wie beispielsweise der Multiplen Sklerose (MS) und Myasthenia Gravis (MG) vor. Der genaue Mechanismus von EBV in diesen autoimmun mediierten Erkrankungen ist noch nicht vollständig geklärt. Um die Rolle des Virus im Pathomechanismus dieser Erkrankungen zu verstehen, führten wir drei Studien durch, die den Zusammenhang zwischen EBV Infektion und Autoimmunität untersuchen sollten. Hierbei konzentrierten wir uns auf die drei Erkrankungen IM, MS und MG. Grundlage der ersten Studie war die Hypothese, dass während einer fulminanten IM- Infektion anhaltende autoreaktive humorale und zelluläre Reaktionen generiert werden, die zu toimmunpathologien im späteren Leben führen. Wir fanden eine erhöhte humorale Immunantwort, die mit der EBV-Viruslast positiv korrelierte. Dies legt nahe, dass die mit EBV in Verbindung stehende humorale Antwort zur Immunpathologie im späteren Leben beiträgt. Als nächstes untersuchten wir, ob eine Behandlung mit IFN-β bei MS-Patienten die EBV-spezifische Immunantwort beeinflusst. Wir fanden in MS-Patienten eine reduzierte zelluläre Antwort auf EBNA1, jedoch keine Veränderung der Immunantwort auf andere EBV Antigene. Dies ist ein Indiz dafür, dass EBNA1-spezifische T-Zellen in der Pathogenese der MS eine Rolle spielen. Unsere dritte Studie hatte zum Ziel, die Kontroverse um den Zusammenhang von EBV und MG zu klären. Trotz Berichten über aktive EBV-Infektionen im Thymus von MG Patienten konnten wir nur geringe Anzeichen von EBV-Infektionen auf DNA- und Proteinlevel finden. Folglich konnten wir nicht bestätigen, dass eine aktive EBV-Infektion in der Pathogenese der MG eine Rolle spielt. Trotz zahlreicher Krankheiten, die mit EBV assoziiert sind, gibt es derzeit keine Behandlung, welche auf latente EBV-Infektionen abzielt. In der letzten in dieser Arbeit beschriebenen Studie lag der Fokus auf EBNA1, welches während der latenten Virusphase exprimiert wird. Mit einem Hochdurchsatz-Screening identifizierten wir die Substanz Tiloron als einen Inhibitor von EBNA1-Funktionen, der die Erhaltung und Replikation des Virus während EBV-Infektionen reduziert. Die Substanz zeigte zytotoxische und antiproliferative Effekte auf EBV-positive, jedoch nur in geringem Maß auf EBV-negative Lymphom- Zelllinien. Des Weiteren führte Tiloron zu reduzierter EBV-assoziierter Tumorlast in immungeschwächten Mäusen und wenig reduzierter EBV-Viruslast in EBV-infizierten Mäusen mit einem humanen Immunsystem. Tiloron hat vielversprechendes Potential, nicht nur im Hinblick auf eine Behandlung von EBV-Infektionen, sondern auch im Hinblick auf EBV-assoziierte Erkrankungen. Zusammenfassend bekräftigen unsere Beobachtungen, dass es bei IM-Patienten und bei IFN- β behandelten MS-Patienten eine Assoziation von EBV und Autoimmunität gibt. Unsere Daten betreffend autoreaktiven poly-spezifischen humoralen Immunantworten während der IM deuten darauf hin, dass EBV bereits vor dem Krankheitsausbruch eine Rolle spielt. Das Hochdurchsatz-Screening hat eine Substanz identifiziert, die das Potential hat, bei latenten EBV-Infektionen und EBV-assoziierten Krankheiten eingesetzt zu werden. Mitte der 80er-Jahre nannte de-Thé EBV den „Stein von Rosette für Verständnis von der Rolle von Viren in menschlicher Karziogenese“ [2]. Mit den in dieser Doktorarbeit gewonnen Erkenntnissen glauben wir einen Schritt näher an die Entschlüsselung vom Stein von Rosette gekommen zu sein im Bereich von virus-assoziierten Autoimmunerkrankungen und deren Behandlungen

Linking the ovarian cancer transcriptome and immunome

<p>Abstract</p> <p>Background</p> <p>Autoantigens have been reported in a variety of tumors, providing insight into the interplay between malignancies and the immune response, and also giving rise to novel diagnostic and therapeutic concepts. Why certain tumor-associated proteins induce an immune response remains largely elusive.</p> <p>Results</p> <p>This paper analyzes the proposed link between increased abundance of a protein in cancerous tissue and the increased potential of the protein for induction of a humoral immune response, using ovarian cancer as an example. Public domain data sources on differential gene expression and on autoantigens associated with this malignancy were extracted and compared, using bioinformatics analysis, on the levels of individual genes and proteins, transcriptional coregulation, joint functional pathways, and shared protein-protein interaction networks. Finally, a selected list of ovarian cancer-associated, differentially regulated proteins was tested experimentally for reactivity with antibodies prevalent in sera of ovarian cancer patients.</p> <p>Genes reported as showing differential expression in ovarian cancer exhibited only minor overlap with the public domain list of ovarian cancer autoantigens. However, experimental screening for antibodies directed against antigenic determinants from ovarian cancer-associated proteins yielded clear reactions with sera.</p> <p>Conclusion</p> <p>A link between tumor protein abundance and the likelihood of induction of a humoral immune response in ovarian cancer appears evident.</p