| 1. |
- Carlsson, Hanna, 1978-
(author)
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Laboratory methods for investigation of the immunological orchestra in response to pathogens
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Laboratory methods used for investigation of immune response often involve collection of whole blood and analysis of different biomarkers in blood components or generated from pathogen stimulation of whole blood or peripheral blood mononuclear cells (PBMC). Methods used to measure biomarkers are for example enzyme-linked immunosorbent assay (ELISA) which measures one biomarker at a time or multiplex assays for example x-unknown, multi-analyte profiling (xMAP) by Luminex or proximity extension assay (PEA), which can measure up to just over 3000 biomarkers at a time. Analysis of one biomarker at a time are time consuming, costly, and dependent of a large sample size to enable repeated measurements of different analytes. Therefore, multiplex assays that are time saving, more cost effective and measures multiple bi-omarkers at once in a small sample can be applied. The aim of this thesis was to evaluate multiplex laboratory methods for investigation of the immunological orchestra in response to Borrelia infection and influenza immunisation and if possible, further characterize individuals with different clinical outcomes or serological response, respectively. In our studies (paper I-III) we included 1113 blood donors of which 66 were found to previously have had a subclinical borreliosis (defined as presence of Borrelia-specific antibodies without recall of previous Lyme borreliosis), of the 66 individuals 60 were available for participation. We also included 22 patients previously diagnosed with Lyme neuroborreliosis (LNB). In paper IV we included in total 73 individuals consisting of healthcare workers and patients attending seasonal influenza vaccination. We applied whole blood, PBMC and plasma stimulations and measured a range of cytokines, chemokines and complement factors with ELISA, nephelometry, xMAP and PEA. Our results show that subclinical Lyme borreliosis (SB) individuals display the following pattern, low age, male sex, low amount of secreted interleukin (IL)-17, CCL20 and higher secretion of IL-10 by PBMCs stimulated three days with Borrelia garinii compared to patients with previous Lyme neuroborreliosis (LNB). The subclinical individuals also show higher activation of the complement system in response to Borrelia afzelii. We performed multiplex analysis of complement factors in attempt to further characterize our SB individuals and LNB patient but found the results to deviate largely from reference values retrieved with other standardized methods. This highlights the importance of critical review of generated results from all form of assays. To investigate immune responses after influenza immunisation and further characterize serological responders and nonresponders we included measurement of influenza-specific antibodies and total immunoglobulins (Ig) in blood serum, influenza-specific mucosal IgA (nasal-swabs) and cell-mediated immune response in supernatants from PBMCs stimulated with influenza vaccine using PEA. We found the serological responders to be characterised by lower levels of total IgM, Granzyme B (GZMB) and IL-12 together with higher levels of CXCL13 compared with nonresponders. To conclude, xMAP and PEA are two valuable methods that can be applied together with multivariate statistical methods in the investigation of both innate and adaptive immunity characteristics and association to clinical outcome or serological response after Borrelia infection and influenza immunisation, respectively.
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| 2. |
- Che, Karlhans Fru
(author)
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Immunomodulatory Effects of Human ImmunodeficiencyVirus (HIV-1) on Dendritic Cell and T cell Responses : Studies of HIV-1 effects on Dendritic cell functionality reflected in primed T cells
- 2011
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Doctoral thesis (other academic/artistic)abstract
- The human immunodeficiency virus (HIV)-1 is the causative agent of acquired immune deficiency syndrome (AIDS) worldwide. Till date there are no vaccines or cure for this infection as the virus has adapted myriad ways to remain persistent in the host where it causes severe damage to the immune system. Both humoral and cellular immune responses are mounted against HIV-1 during the initial phase of infection but fail to control viral replication as these responses are severely depleted during disease progression. Of great importance in HIV-1 research today is the in depth understanding of the types of immune responses elicited, the mechanisms behind their decline and how these responses can be maintained overtime.The focus of this thesis was to examine the possibility of priming HIV-1 specific T cell responses in vitro from whole viral particles and in detail, scrutinize the type of T cell responses and epitope specificities generated. Next was to investigate in vitro the factors responsible for impaired immune responses in HIV-1 infected individuals. We were also interested in understanding the underlying mechanisms through which HIV-1 initiate suppression of T cell functionality.Results showed that using HIV-1 pulsed monocyte derived dendritic cells (DCs), we were able to prime HIV-1 specific CD4+ and CD8+ T cells from naïve T cells in vitro. The epitopes primed in vitro were located within the HIV-1 envelope, gag, and pol proteins and were confirmed ex vivo to exist in acute and chronically infected individuals. We established that many of the novel CD4+ T cell epitopes primed in vitro also existed in vivo in HIV-1 infected individuals during acute infection. These responses declined/disappeared early on, which is in line with HIV-1 preferential infection of HIV-1 specific CD4+ T cells.Besides declining HIV-1 specific T cell responses, many HIV-1 infected individuals also have impaired T cell functionality. We established that one reason behind the decline and impairment in immune responses was the increased expression of inhibitory molecules PD-1, CTLA-4, and TRAIL on HIV-1 primed T cells. These T cells had the capacity to suppress new responses in a cell-cell contact dependent manner. The ability of the HIV-1 primed T cells to proliferate was severely impaired and this condition was reversed after a combined blockade of PD-1, CTLA-4 and TRAIL. Furthermore, more inhibitory molecules TIM-3, LAG-3, CD160, BLIMP-1, and FOXP3 were also found increased at both gene and protein levels on HIV-1 primed T cells. Additionally, we showed decreased levels of functional cytokines IL-2, IFN-γ and TNF-α, and the cytolytic proteins perforin and granzyme in DC T cell priming cocultures containing HIV-1. This could be as a result of the decreased T cell activation or impaired production by T cells. The mechanisms responsible for the elevated levels of inhibitory molecules emanated mainly from the P38MAPK/STAT3 pathways. Blockade of these pathways in both allogeneic and autologous DC-T cell assays significantly suppressed expression of inhibitory molecules and subsequently rescued T cell proliferation.In conclusion, HIV-1 pulsed DCs have the capacity to prime HIV-1 specific responses in vitro that do exist in HIV-1 infected individuals and we found evidence that many of these responses were eliminated rapidly in HIV-1 infected individuals. HIV-1 triggers through P38MAPK/STAT3 pathway the synthesis of inhibitory molecules, namely CTLA-4, PD-1, TRAIL, TIM-3, LAG-3, CD160, and suppression associated transcription factors FOXP3, BLIMP-1 and DTX1. This is followed by decreased T cell proliferation and functionality which are much needed to control viral replication.
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| 3. |
- Kindberg, Elin, 1981-
(author)
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Host genetic risk factors to viral diseases - a double-edged sword : Studies of norovirus and tick-borne encephalitis virus
- 2010
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Doctoral thesis (other academic/artistic)abstract
- It is today well known that the outcome of a certain infection depends on factors of both the host and the pathogen. Studies of host genetic susceptibility to infectious diseases aim to increase the understanding of why some individuals are more susceptible than others, to a certain infection. Knowledge of genetic susceptibility to a viral disease may be used in development of new therapeutic means, and also to recognize individuals who are at increased risk of severe symptoms if infected with a pathogen. It seems however that a risk factor for one disease may play a protective role in another situation; like a double-edged sword.In this thesis I have studied genetic factors affecting susceptibility to norovirus (NoV) and factors affecting the risk of developing tick-borne encephalitis (TBE) after infection with TBE virus (TBEV). NoV is the cause of the “winter vomiting disease”, affecting millions of people every year, and causing up to 200,000 fatalities among children in developing countries, each year. It is today recognized that the secretor status of an individual, i.e. the ability to express ABO blood groups and related antigens, in secretions and on mucosa, affect the risk of being infected by NoV. By studying authentic NoV outbreaks in Denmark, Spain and Sweden and by comparing the secretor status of affected and unaffected individuals we were able to confirm that secretor status have indeed great impact on susceptibility to some NoV strains, but also that there are strains circulating, which infect individuals regardless of secretor status.TBEV is endemic in many parts of Europe and Asia but studies have shown that 70-95% of all infections are asymptomatic or sub-clinical. Some individuals do however develop TBE, a severe disease including meningitis or encephalitis with or without myelitis. Also, many patients suffer from long-time sequelae and TBEV infections may in worst case be fatal. The reason for difference in disease outcome is not known and we have chosen to study if genetic factors affecting the immune response may play a role in disease outcome. To do this we used a prospectively collected Lithuanian material with samples from patients with TBE, AME (aseptic meningoencephalitis) and matched healthy controls. So far we have found that a deletion in chemokine receptor 5 (CCR5), a gene encoding a receptor involved in cell migration, is a risk factor for developing disease. We have also data showing that toll-like receptor 3 (TLR3), a receptor recognizing double stranded RNA (dsRNA), which is a product of TBEV replication, may instead of being protective increase the risk of TBE.
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| 4. |
- Tjomsland, Veronica
(author)
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Complement activation - good or evil in HIV-1 infection? : interaction of free and complement opsonized HIV-1 with monocyte derived dendritic cells and immune cells in the cervical mucosa
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Worldwide, the heterosexual route is the most common mode of sexual transmission of HIV-1 and women are particularly susceptible to this infection. After penetration of the mucosal epithelium HIV-1 interacts with potential target cells, i.e. dendritic cells (DCs) and CD4+ T cells. The complement system, a key component of the innate immune system, is immediately activated by HIV-1 in vivo. However, HIV-1 can resist complement mediated lysis and become coated with complement fragments and this opsonization influences the viral interaction with immune cells. The DCs are the most potent antigen presenting cell. This cell effectively links the innate recognition of viruses to the generation of an adaptive immune response. However, HIV-1 exploits the function of the DCs to facilitate viral spread and infection. HIV-1 interacts with a range of receptors expressed by the DCs including C-type lectins, integrins and complement receptors (CRs). The uptake of virions by DCs leads to their activation and migration to the lymph nodes. At this site DCs present HIV-1 derived antigen on MHC class I and II molecules and trigger an HIV-1 specific T cell response. The interplay between the virus and the DCs is complex and the initial receptor binding may affect antigen uptake, infection, and antigen presentation.The fundamental questions of this thesis are the following: How is free and opsonized HIV-1 internalized, processed, and presented on MHC class I and II molecules by DCs and how do free and opsonized HIV-1 particles interact with immune cells in the cervical mucosa?Our results indicate that opsonization of HIV-1 plays a critical role in the interaction with immune cells. Complement opsonization of HIV-1 (C-HIV) significantly enhanced the internalization by the DCs compared to free HIV (F-HIV). Both C-HIV and F-HIV interacted with the CD4 receptor, C-type lectins and integrins. In addition, opsonization of HIV-1 favored an MHC class I presentation by DCs compared to F-HIV. However, the endocytic receptors macrophage mannose receptor, β7 integrin, and CR3 guided the antigens to different compartments with distinct properties and efficiencies for degradation and MHC class I and II presentation of viral antigens. MHC class I presentation of F-HIV and C-HIV was dependent of viral fusion in a CD4/coreceptor dependent manner. Moreover, MHC class II presentation of antigens derived from HIV-1 required endocytosis and proteolysis in acidified compartments. HIV-1 infection of cervical mucosa immune cells and tissue was assessed in a cervical tissue explant model. C-HIV significantly enhanced infection of DCs compared to F-HIV, whereas C-HIV decreased the infection of CD4+ T cells. Blocking the viral use of integrins in the cervical tissue explants significantly decreased the HIV-1 infection of both emigrating DCs and CD4+ T cells and the establishment of founder populations in these tissues. This thesis work has brought forward new facts that can be used to facilitate the development of an effective vaccine or microbicide.
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| 5. |
- Svanberg, Cecilia, 1991-
(author)
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HIV-1 Exploitation of Dendritic Cell Functionality and Initial Responses in Mucosal Tissues : Elucidation of Influence of HIV-1 Complement Opsonization, and HIV-1-HSV-2 Co-infection
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Human immunodeficiency virus (HIV)-1 is transmitted between individuals via sexual intercourse or via blood products. To date there are 38 million people living with chronic HIV infection and around 1.5 million yearly acquire a new infection. HIV is known to have detrimental effects on the immune system and its cell function and leads to the development of acquired immune deficiency syndrome (AIDS). AIDS is characterized by opportunistic infections often fatal to the individual. Mucosal tissues, which are the main site of HIV-1 infection, consist of a complex network of different cell types building up a barrier against the outside world. In the mucosa there are multiple immune cells and one of them is the dendritic cells (DCs), a professional antigen presenting cell needed for priming of adaptive T cell responses. DCs are rarely productively infected with HIV-1 but the virus can modulate the DC phenotype and function by mere exposure to the virus. There are many factors that influence viral transmission including prevalent inflammatory conditions or infections in the genital tract. For instance, a newly acquired herpes simplex virus-2 (HSV-2) infection increases the risk of transmission at HIV-1 exposure by four times. A vital part of the innate immune defense is the complement system, which consists of proteins found in all body fluids. Normally during complement activation, it leads to lysis of pathogens, but HIV-1 can evade this process. This is achieved by the incorporated complement regulatory proteins in the viral plasma membrane leading to accumulation of opsonizing complement fragments on the HIV-1 particles. Our aim with these studies was to deepen the understanding of the HIV-1 exploitation of DC function and elucidate the initial effects exposure and establishment of HIV-1 infection has on the mucosal tissues and immune cells. In addition, we aimed to elucidate the effects HSV-2 exerts on the HIV-1-infection of DCs and how viral complement opsonization affects the viral infection and activation of immune responses. In Paper I we found that coinfection of monocyte derived DCs (MDDCs) with HIV-1 and HSV-2 decreases the amount of the HIV-1 restriction factors SAMHD1 and TREX1 in MDDCs, leading to increased productive HIV-1 infection. In Paper II and III we found that HIV-1 utilizes the complement system to induce higher productive infection of the colorectal and cervical mucosal DCs. The different forms of HIV, free or complement opsonized, had distinct effects on the immune responses and T cell phenotypes in the tissues, all in favor of HIV-1 establishment and productive infection. In Paper IV, HIV-1 exposed DCs triggered after crosstalk with suppressive T cells a prolonged type I interferon response and an upregulation of coinhibitory molecules on the DCs. The findings in this thesis add to the knowledge of HIV-1 early transmission events, how co-infection modulates DC function, and how the presence of HIV-1 affects the priming of adaptive immune responses.
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| 6. |
- Falkeborn, Tina, 1981-
(author)
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Nasal vaccination using novel mucosal adjuvants : with main focus on influenza A virus
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Influenza viruses have sporadically caused pandemics during the last century, with the most severe occurring in 1918 when the “Spanish flu”, an A/H1N1 influenza virus, passed around the globe killing about 20-100 million people. Today 250 000-500 000 deaths occur annually due to influenza virus or secondary infection after influenza, e.g. pneumonia. Influenza viruses cause severe infections in susceptible age groups like children and elderly and in individuals with impaired immune response due to other medical conditions. The best way to prevent an influenza epidemic is by vaccination. Since the 1950´s we have vaccines against seasonal flu, but vaccine efficacy is not 100 % and there is a need to develop better and more effective vaccines, especially for the risk groups. Since the virus enters the host through the nasal cavity, nasal vaccination is a good approach. By stimulating a mucosal immune response already in the nasal cavity, the goal with nasal vaccination is to stop the virus before it enters the host. Nasal vaccination also reduces the risk of transmission of blood-borne diseases, and is less painful and easier to administer, compared to injectable vaccines.In order to be able to use less immunogenic antigens, like split and subunit antigens, as nasal vaccine components, an adjuvant is needed to enhance the immune response. At the moment there is no licensed mucosal adjuvant for human use. Several studies are ongoing, but it is a complicated and long way to reach the market. In this thesis nasal vaccination with influenza antigen together with the mucosal adjuvant Endocine™ and other mucosal adjuvants has been evaluated. The Endocine™ adjuvant has been shown to be safe and well tolerated in clinical trials. Depending on the pathogen of interest, different approaches are necessary. For HIV, DNA-vaccination has been evaluated together with a plasmid encoding Salmonella typhimurium flagellin C and the mucosal adjuvant N3. The results found in paper I-IV show that by adding adjuvant to the antigen enhances the protective immune response towards the antigen. Enhanced systemic, mucosal and cell-mediated immunity were observed. Hopefully in the future these adjuvants evaluated in this thesis, will be used in vaccines for humans.
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| 7. |
- Ellegård, Rada, 1985-
(author)
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Effects of Complement Opsonization of HIV on Dendritic Cells : and Implications for the Immune Response
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Dendritic cells are key players during HIV pathogenesis, and shape both the immediate immune response at the site of infection as well as directing the adaptive immune response against the virus. HIV has developed a plethora of immune evasion mechanisms that hijack dendritic cell functions, suppressing their ability to mount an accurate immune response and exploiting them for efficient viral transfer to target T cells.To achieve successful replication within dendritic cells without triggering danger signaling, HIV accomplishes a delicate balance where only a low level of transcription can be sustained without triggering antiviral responses that would harm the virus. Here, we describe how the presence of HSV2 coinfection, which is very common in geographic areas with a high HIV prevalence and almost triples the risk of HIV acquisition, alters dendritic cell state to support much higher levels of HIV infection. We found this effect to be mediated by the STING pathway, which is involved in the sensing of DNA in the cell cytosol. STING activation led to an upregulation of factors such as IRF3 and NFkB that can be used for HIV transcription and a degradation of factors that restrict HIV replication.In addition, we describe how HIV exploits the human complement system, a group of proteins that usually help the human body to identify dangerous pathogens while avoiding reaction towards self. HIV can coat itself, i.e. become opsonized, in complement fragments that are typically only present on the body’s own cells, allowing it to activate signaling pathways that are associated with tolerance. Dendritic cells that come into contact with complement opsonized HIV do not mount danger responses, despite the fact that HIV-derived single stranded RNA triggers the pathogen recognition receptor TLR8. The suppression of danger responses is mediated by activation of complement receptor 3, and leads to an increased infection of the dendritic cell and affects its interactions with other immune cells. There is a lack of recruitment of NK cells to the site of infection, and an inhibition of NK cell killing, which plays an important role in the destruction of HIV-infected cells in vivo. T cells primed by dendritic cells exposed to complement opsonized HIV have a lower ability to develop towards effector phenotype, and have an increased expression of the markers PD1, TIM3 and LAG3 which are associated with T cell dysfunction and exhaustion. In addition, T cells primed by these dendritic cells in the presence of NK cells upregulate markers CD38, CXCR3 and CCR4, which have been linked to an increased susceptibility to HIV infection.In summary, we add to the current knowledge on HIV immune evasion mechanisms that allow the virus to establish infection, as well as describing mechanisms that govern whether dendritic cells mount danger signaling and an immune response or not.
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