From predicting to analyzing {HIV}-1 resistance to broadly neutralizing antibodies

Treatment with broadly neutralizing antibodies (bNAbs) has recently proven effective against HIV-1 infections in humanized mice, non-human primates, and humans. For optimal treatment, susceptibility of the patient’s viral strains to a particular bNAb has to be ensured. Since no computational approaches are so far available, susceptibility can only be tested in expensive and time-consuming neutralization experiments. Here, we present well-performing computational models (AUC up to 0.84) that can predict HIV-1 resistance to bNAbs given the envelope sequence of the virus. Having learnt important binding sites of the bNAbs from the envelope sequence, the models are also biologically meaningful and useful for epitope recognition. Additional to the prediction result, we provide a motif logo that displays the contribution of the pivotal residues of the test sequence to the prediction. As our prediction models are based on non-linear kernels, we introduce a new visualization technique to improve the model interpretability. Moreover, we confirmed previous experimental findings that there is a trend towards antibody resistance for the subtype B population of the virus. While previous experiments considered rather small and selected cohorts, we were able to show a similar trend for the global HIV-1 population comprising all major subtypes by predicting the neutralization sensitivity for around 36,000 HIV-1 sequences- a scale-up which is very difficult to achieve in an experimental setting

Predicting and analyzing HIV-1 adaptation to broadly neutralizing antibodies and the host immune system using machine learning

Thanks to its extraordinarily high mutation and replication rate, the human immunodeficiency virus type 1 (HIV-1) is able to rapidly adapt to the selection pressure imposed by the host immune system or antiretroviral drug exposure. With neither a cure nor a vaccine at hand, viral control is a major pillar in the combat of the HIV-1 pandemic. Without drug exposure, interindividual differences in viral control are partly influenced by host genetic factors like the human leukocyte antigen (HLA) system, and viral genetic factors like the predominant coreceptor usage of the virus. Thus, a close monitoring of the viral population within the patients and adjustments in the treatment regimens, as well as a continuous development of new drug components are indispensable measures to counteract the emergence of viral escape variants. To this end, a fast and accurate determination of the viral adaptation is essential for a successful treatment. This thesis is based upon four studies that aim to develop and apply statistical learning methods to (i) predict adaptation of the virus to broadly neutralizing antibodies (bNAbs), a promising new treatment option, (ii) advance antibody-mediated immunotherapy for clinical usage, and (iii) predict viral adaptation to the HLA system to further understand the switch in HIV-1 coreceptor usage. In total, this thesis comprises several statistical learning approaches to predict HIV-1 adaptation, thereby, enabling a better control of HIV-1 infections.Dank seiner außergewöhnlich hohen Mutations- und Replikationsrate ist das humane Immundefizienzvirus Typ 1 (HIV-1) in der Lage sich schnell an den vom Immunsystem des Wirtes oder durch die antiretrovirale Arzneimittelexposition ausgeübten Selektionsdruck anzupassen. Da weder ein Heilmittel noch ein Impfstoff verfügbar sind, ist die Viruskontrolle eine wichtige Säule im Kampf gegen die HIV-1-Pandemie. Ohne Arzneimittelexposition werden interindividuelle Unterschiede in der Viruskontrolle teilweise durch genetische Faktoren des Wirts wie das humane Leukozytenantigensystem (HLA) und virale genetische Faktoren wie die vorherrschende Korezeptornutzung des Virus beeinflusst. Eine genaue Überwachung der Viruspopulation innerhalb des Patienten, gegebenfalls Anpassungen der Behandlungsschemata sowie eine kontinuierliche Entwicklung neuer Wirkstoffkomponenten sind daher unerlässliche Maßnahmen, um dem Auftreten viraler Fluchtvarianten entgegenzuwirken. Für eine erfolgreiche Behandlung ist eine schnelle und genaue Bestimmung der Anpassung einer Variante essentiell. Die Thesis basiert auf vier Studien, deren Ziel es ist statistische Lernverfahren zu entwickeln und anzuwenden, um (1) die Anpassung von HIV-1 an breit neutralisierende Antikörper, eine neuartige vielversprechende Therapieoption, vorherzusagen, (2) den Einsatz von Antikörper-basierte Immuntherapien für den klinischen Einsatz voranzutreiben, und (3) die virale Anpassung von HIV-1 an das HLA-System vorherzusagen, um den Wechsel der HIV-1 Korezeptornutzung besser zu verstehen. Zusammenfassend umfasst diese Thesis mehrere statistische Lernverfahrenansätze, um HIV Anpassung vorherzusagen, wodurch eine bessere Kontrolle von HIV-1 Infektionen ermöglicht wird

A Novel Methodology for Isolating Broadly Neutralizing HIV-1 Human Monoclonal Antibodies

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-53. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

Evaluations Of Severe Acute Respiratory Syndrome Coronavirus Therapeutics And A Viral Capacity For Plasticity And Escape.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emerged in 2002/2003, causing the deaths of almost a tenth of the 8000 individuals infected worldwide before it was controlled by public health measures. While the 2003 epidemic strain is likely extinct, the importance of coronaviruses as emergent zoonotic viruses was again realized with the emergence of a novel human coronavirus in Saudi Arabia in 2012. Despite a decade of research on SARS-CoV no approved vaccine or therapeutic yet exists, and development of broadly neutralizing and effective therapeutics for coronaviruses remains a priority. Neutralizing antibodies targeting the Spike glycoprotein (S) are both necessary and sufficient for protection against SARS-CoV, but the high genetic diversity and mutability of SARS-CoV in natural infections presents a challenge to both vaccine- and antibody-based therapeutics. Thus, an effective SARS-CoV therapeutic should provide S-specific immunity that is nonetheless broad enough to counter heterologous and derivative S variants. This work was designed to assess immunization strategies towards SARS-CoV, to explore the plasticity and neutralization networks of the Spike glycoprotein, and to assess the utility of molecular models to predict host range and antibody neutralization. In the first study we explored the limitations of a doubly inactivated SARS-CoV vaccine, identifying a vaccine-induced immunopathology and emphasizing the importance of rigorous challenge viruses and animal models that accurately recapitulate age-associated lung pathology. Second, in two collaborative studies we assessed multi-generational monoclonal antibodies designed to be broadly neutralizing or escape resistant, and extended our characterization of the Spike receptor binding domain (RBD) as a highly plastic antiviral target. Finally, we characterized ten recombinant Combinatorial Escape Viruses (CEVs) engineered from a database of antibody escape substitutions in the RBD. These CEVs were designed to assess the plasticity of the S-RBD, the utility of predictive modeling, and the neutralization networks across the RBD. The tools developed this study will assist in the development of predictive models and standardized platforms for combination monoclonal antibody immunotherapies for emergent viruses. These studies of SARS-CoV have extended our understanding of a key neutralizing target and have provided a valuable foundation for the rapid characterization of novel coronaviruses and potential therapeutics.Doctor of Philosoph

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs–specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades

HIV infection in Angola : Molecular epidemiology, diagnosis and antibody neutralization

The UNAIDS fast-track goals established the need to reduce 75% of new HIV cases and deaths by 2020 and 90% by 2030, compared to 2010 [1]. However, it is estimated that no country in sub-Saharan Africa has reached the target of 75% reduction in new cases of HIV infection [2]. HIV-1 incidence in Angola increased 61.2% from 2010 to 2018 and the prevalence is now stable with 340,000 people living with HIV [2, 3]. Women and children are the most affected populations [3]. The aims of this thesis were: 1) to get a better understanding of the HIV-1 diversity, transmission dynamics and transmitted drug resistance (TDR) in Angola, 2) improve the early infant diagnosis (EID) in Angola and 3) characterize the neutralizing antibody responses against HIV-1 and assess the possible viral and host factors associated with it. The HIV epidemics in Angola is peculiar, since highly divergent forms of the virus circulates in the population. In this context our first objective was to assess HIV-1 diversity, transmission dynamics and prevalence of transmitted drug resistance (TDR) in Angola in 2009, in 139 drug naïve HIV-1 infected individuals and compare the results before ART scale-up (chapter 2). We saw an increase in genetic diversity between 2001 and 2009, with the prevalence of subtype A decreasing significantly while the prevalence of unique recombinant forms (URFs) increased 2-fold. Also, local U/H recombinants were newly identified. TDR mutation K103N was found in one (0.7%) patient. Overall, transmission of drug resistant strains was still negligible in Luanda in 2009 and the emergence of new URFs are consistent with a rising HIV-1 epidemics. Our second objective was to develop and validate a sensitive, simple and cheap qualitative proviral DNA PCR-based assay for early infant diagnosis (EID) in HIV-1-exposed infants (n=139) using dried blood spots (DBS) (chapter 3). We were able to successfully validate the assay, the limit of detection (LOD) using several integrase recombinant plasmids was 14 copies and clinical sensitivity and specificity were high. The percentage of HIV-1 mother-to-child-transmission (MTCT) between January 2012 and October 2014 was only 2.2%. In chapter 4 we performed the first detailed characterization of the neutralizing antibody response in 322 Angolan HIV-1 infected patients and identified its determinants. Remarkably, 56% of the individuals had broad cross-neutralizing activity. Also, cross-clade neutralization was positively associated with subtype C infection and negatively associated with CD4 counts and antibody binding titers against envelope C2V3C3 region was a good indicator of neutralization breadth and potency. In chapter 4 we concluded that development of broad and elite antibody neutralization against HIV-1 requires long-term and low-level envelope V3C3 stimulation from highly diverse subtype C isolates

Elicitation of broadly neutralizing HIV-1 antibodies by guiding the immune responses using primary and secondary immunogens

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-44. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

Characterizing the impact of the mutational landscape of SARS-CoV-2 on epitope presentation and CTL

La pandémie actuelle de COVID-19, causée par le coronavirus 2 du syndrome respiratoire aigu sévère (SRAS-CoV-2), a entraîné plus de 6 millions de décès et près de 680 millions de cas confirmés dans le monde. Depuis l'émergence du virus en décembre 2019, beaucoup d’efforts de recherche mondiaux ont visé à étudier la relation entre le SRAS-CoV-2 et l'immunité adaptative à médiation cellulaire. La caractérisation des réponses immunitaires à base de lymphocytes T CD4+ et CD8+ contre le SRAS-CoV-2 dans le contexte de mutations virales est d'une pertinence immédiate pour l’approfondissement de nos connaissances concernant l'immunité adaptative envers un virus en évolution, ainsi que l'amélioration de vaccins. Dans cette thèse, je passerai en revue les découvertes actuelles concernant la biologie du SRAS-CoV-2 et sa relation avec le système immunitaire adaptatif humain. Je discuterai ensuite les divers mécanismes par lesquels le SRAS-CoV-2, ainsi que d'autres virus, se sont avérés échapper l’immunité adaptative humoral et cellulaire. Enfin, je présenterai mes contributions à la compréhension du paysage mutationnel global du SRAS-CoV-2 et de sa capacité à échapper à la reconnaissance par les lymphocytes T CD8+. Dans ce travail, j'ai observé que le paysage mutationnel global du SRAS-CoV-2 était régi par des biais de mutation au cours de la première année de la pandémie, le plus répandu d’entre eux conduisant à la suppression de la proline. Il a ensuite été prédit que cette élimination globale de la proline conduirait à la perte d’épitopes reconnues par les cellules T CD8+ d'une manière dépendante sur les super-types HLA, avec la perte d'épitopes survenant préférentiellement dans le contexte du super-type HLA-B7. Le modèle développé dans ce travail propose un lien entre les biais mutationnels globaux du SRAS-CoV-2, les allèles HLA et l'évasion des lymphocytes T. Ce travail crée un cadre pour anticiper l'impact des variantes existantes et émergentes du SRAS-CoV- 2 envers la réponse immunitaire à base de lymphocytes T CD8+.The current COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to upwards of 6 million deaths and nearly 680 million confirmed cases worldwide. Since the emergence of the virus in December 2019, astounding global research efforts have been aimed at investigating the relationship between SARS-CoV-2 and cell-mediated adaptive immunity. Characterizing CD4+ and CD8+ T Lymphocyte responses to SARS-CoV-2 in the context of viral mutations is of immediate relevance to understanding the breadth of a population’s adaptive immunity to an evolving virus and is central to the improving existing vaccines. In this thesis, I will review all present findings pertaining to the biology of SARS-CoV-2 and its relationship with the human adaptive immune system. I will then discuss the various mechanisms by which SARS-CoV-2, along with other viruses, have been found to evade the various arms of the adaptive immune system. Finally, I will present my contributions to the understanding of the global mutational landscape of SARS-CoV-2 and its ability to evade recognition by CD8+ T lymphocytes. By investigating over 300,000 SARS-CoV-2 genomic sequences, I observed that the mutational landscape of SARS-CoV-2 was governed by mutation biases during the first year of the pandemic, with the most prevalent bias leading to the removal of proline. The observed global removal of Proline was predicted to lead to the loss of CD8+ T cell epitopes in an HLA-supertype-dependent manner, with the loss of epitopes occurring preferentially in the context of the HLA-B7 supertype. The model developed proposes a link between SARS-CoV-2 global mutational biases, HLA alleles and T cell evasion. This work creates a framework to anticipate the population-specific impact of existing and emerging SARS-CoV-2 variants on CD8+ T cell-based immunity