Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

The enzyme-linked immunospot (ELISPOT) assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures) or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT) or after their expansion during a 10-day culture (cultured ELISPOT). Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field

Functionalized Plasmonic Nanostructures for Ultrasensitive Single Cell Analysis

Ultrasensitive detection and quantification of soluble, secreted and cell surface-bound proteins is critical for advancing our understanding of cellular systems, enabling effective drug development, novel therapies, and bio-diagnostics. However, exiting technologies are largely limited by their sensitivity, making the detection and quantification of low-abundant proteins extremely challenging. This forms a major barrier in various fields of biology and biomedical sciences. In this work, we introduce novel cellular analysis methodologies based on plasmon-enhanced fluorescence for analyzing cell structure and probing surface and secreted proteins from cells. In the first part, we introduce plasmon-enhanced expansion microscopy and demonstrate the effectiveness of employing an ultrabright plasmonic nanolabel in probing hippocampal neurons and quantifying the morphological markers at high resolution. In the second part of this thesis, we introduce plasmon-enhanced FluoroDOT assay for ultrasensitive detection of cell secreted proteins. The plasmonic nanolabels enabled significantly improved signal-to-noise ratio compared to conventional fluorophores, therefore enabling detection and quantification of cell secreted proteins at extremely low concentrations of chemical or biological stimuli. In the third part, we establish plasmon-enhanced flow cytometry as a novel methodology to probe and analyze cellular surface proteins, enhancing the sensitivity of the approach in delineating cell populations with different protein levels. Overall, we establish the proof-of-concept for various plasmon-enhanced cellular analysis and biodetection methods that can be potentially useful in advancing the field of biological and biomedical sciences

Enumeration of Functional T-Cell Subsets by Fluorescence-Immunospot Defines Signatures of Pathogen Burden in Tuberculosis

IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation

Cell Detection by Functional Inverse Diffusion and Non-negative Group Sparsity−-Part I: Modeling and Inverse Problems

In this two-part paper, we present a novel framework and methodology to analyze data from certain image-based biochemical assays, e.g., ELISPOT and Fluorospot assays. In this first part, we start by presenting a physical partial differential equations (PDE) model up to image acquisition for these biochemical assays. Then, we use the PDEs' Green function to derive a novel parametrization of the acquired images. This parametrization allows us to propose a functional optimization problem to address inverse diffusion. In particular, we propose a non-negative group-sparsity regularized optimization problem with the goal of localizing and characterizing the biological cells involved in the said assays. We continue by proposing a suitable discretization scheme that enables both the generation of synthetic data and implementable algorithms to address inverse diffusion. We end Part I by providing a preliminary comparison between the results of our methodology and an expert human labeler on real data. Part II is devoted to providing an accelerated proximal gradient algorithm to solve the proposed problem and to the empirical validation of our methodology.Comment: published, 15 page

Methods for studying memory B-cell immunity against malaria

Plasmodium falciparum malaria remains one of the world’s deadliest infectious diseases and the search for an effective vaccine is highly warranted. Memory B cells (MBCs) and the antibodies they produce, once activated, is believed to play an important role in the protective immunity against malaria, but the mechanism of acquiring and maintaining these cells is poorly understood. New and sensitive tools able of gathering detailed information regarding the development and maintenance of antigen-specific MBCs could increase the understanding of protective immunity but also be used for the evaluation of new vaccines. In Study I, we developed the reversed B-cell FluoroSpot assay, a new assay format based on an established technique for single-cell analysis. Using hybridomas and splenocytes from immunized mice together with a tag/anti-tag approach for detection, we showed proof-of-principle that the assay could be used for multiplex analysis of single B cells specific to four different antigens simultaneously, as well as detecting B cells displaying cross-reactivity against antigen variants. In Study II, we adapted the assay for studies on humans and measured MBC responses against hepatitis B virus, tetanus toxoid and cytomegalovirus. We also measured MBC frequencies before and after vaccination against hepatitis B and used new FluoroSpot reader functions to assess spot volume. We showed that the assay could be used to detect B cells against all of the antigens simultaneously and also changes in MBC frequencies and spot volume before and after vaccination. In Study III, we adapted the multiplex assay further for studies on P. falciparum antigen-specific MBCs and used it to study the kinetics of MBC responses in primary infected and previously exposed travelers diagnosed with malaria in Sweden. We showed that primary infected individuals could acquire and maintain P. falciparum-antigen specific MBCs as efficiently as previously exposed individuals during a one year follow up period, but that the maintenance and magnitude of antibody levels in plasma were higher in the previously exposed individuals. In Study IV, we used the assay developed in Study III to analyze P. falciparum antigen-specific MBCs in children living in areas with endemic transmission of malaria in Kenya. We identified that high levels of MBCs against certain P. falciparum antigens were associated with a reduced risk of a subsequent clinical malaria episode, and that proportions of MBCs specific to some, but not all, P. falciparum antigens, increase with age, but also some decrease with cumulative number of infections. We conclude that the multiplex FluoroSpot method developed in this thesis provide insights towards the acquisition and maintenance of P. falciparum malaria-induced MBCs. We believe that the reversed B-cell FluoroSpot assay is a sensitive and highly adaptable method to assess MBC responses against multiple antigens and will be a powerful tool for future studies on protective immunity to malaria, but also other fields of research

ELISpot for measuring human immune responses to vaccines

The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-γ secretion and granzyme B release. Furthermore, ELISpot is adaptable not only to the evaluation of a variety of T-cell functions, but also to B cells and innate immune cells. It is no wonder that ELISpot has evolved from a research tool to a clinical assay. Recent Phase I and II studies of cancer vaccines, tested in a variety of malignancies, have suggested that ELISpot may be a useful biomarker assay to predict clinical benefit after therapeutic immune modulation. This article will discuss the most common applications of ELISpot, overview the efforts that have been undertaken to standardize the assay and apply the method in the analysis of human clinical trials, and describe some important steps in the process of developing a clinical-grade ELISpotope

Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using Elispot Assays: Looking Beyond Interferon-gamma

Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subject's blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach

Adaptive immune responses to tick-borne encephalitis virus and SARS-CoV-2

Tick-borne encephalitis virus (TBEV) and SARS-CoV-2 are two unrelated viruses that currently cause substantially different public health burdens and distinct pathologies in humans. TBEV infection leads to neurological symptoms of varying severity, while SARS-CoV-2 primarily targets the respiratory tract. The aims of this thesis were to estimate the relative level of mortality due to TBE in Sweden (Paper I) and to describe the human adaptive immune responses to TBE vaccination (Paper II), TBEV infection (Paper III), and SARS-CoV-2 infection (Papers IV-V). In Paper I, we measured the standardized mortality ratio (SMR) in TBE patients and found that, compared to a matched control population, TBE patients experience around a four-times higher mortality within 90 days after the diagnosis. Considering that TBE is a vaccinepreventable disease, this finding highlights the need for increased vaccination efforts for people at risk of exposure to TBEV. In Paper II, we assessed memory T cell responses throughout the primary immunization schedule with TBE (three doses within one year). We observed a heterogenous magnitude of memory CD4+ T cell response in the TBE vaccinated individuals, with the highest magnitude after the 2nd dose. Compared to TBE patients, TBE vaccinees had fewer polyfunctional memory CD4+ T cells and lower IFN-g responses. This study suggests that the TBE vaccine elicits a lower quality of CD4+ T cell memory compared to TBE infection and highlights the need for the development of improved TBE vaccines. In Paper III, we assessed the antibody-secreting cell (ASC) responses and TBEV-specific antibody levels in TBE patients at varying timepoints after hospitalization. ASC expansion is typically a hallmark of early B cell responses during acute infections. Compared to dengue patients, who served as a control cohort in this study, low frequencies of ASCs were detected in TBE patients at all four sampling timepoints (i.e., 30 days after hospitalization). In addition, all TBE patients had detectable TBEV-specific IgM and IgG antibody levels throughout the course of the study. These findings indicate that the early B cell response may take place even earlier during TBE, likely before hospitalization. In Papers IV and V, we investigated germinal center activity, ASC responses and antibody levels during the acute SARS-CoV-2 infection in hospitalized COVID-19 patients. We observed an increased germinal center activity and ASC expansion in COVID-19 patients. In Paper V, we subsequently detected polyfunctional memory T cell and memory B cell responses in previously hospitalized recovered COVID-19 patients at 5 and 9 months after symptom onset. This finding indicates that immunological memory to SARS-CoV-2 persists for at least up to 9 months regardless of COVID-19 severity at hospitalization. In conclusion, this thesis contributes to the understanding of TBEV and SARS-CoV-2 infections, particularly in relation to the adaptive human immune responses to these viruses

Cell Detection by Functional Inverse Diffusion and Non-negative Group Sparsity−-Part II: Proximal Optimization and Performance Evaluation

In this two-part paper, we present a novel framework and methodology to analyze data from certain image-based biochemical assays, e.g., ELISPOT and Fluorospot assays. In this second part, we focus on our algorithmic contributions. We provide an algorithm for functional inverse diffusion that solves the variational problem we posed in Part I. As part of the derivation of this algorithm, we present the proximal operator for the non-negative group-sparsity regularizer, which is a novel result that is of interest in itself, also in comparison to previous results on the proximal operator of a sum of functions. We then present a discretized approximated implementation of our algorithm and evaluate it both in terms of operational cell-detection metrics and in terms of distributional optimal-transport metrics.Comment: published, 16 page