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- Carlsson, Hanna, 1978-
(författare)
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Laboratory methods for investigation of the immunological orchestra in response to pathogens
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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. |
- Cardell, Kristina, 1964-
(författare)
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Studies on Hepatitis B Vaccination and Factors Associated with the Vaccine Response
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis B virus causes liver disease and up to 2 billion people have been in contact with the virus world wide. It can cause both acute and chronic disease. The routes for transmission are through blood, mother to infant at time of delivery and sexually. Chronic hepatitis B infection is a risk factor for development of liver cirrhosis and hepatocellular carcinoma. Prevention of hepatitis B virus infection is highly desirable. Since the early1980s hepatitis B vaccine has been available. It can effectively prevent the disease and has been found to be safe. The World Health Organisation, WHO, has recommended all countries to implement the vaccine in their children’s vaccination programmes and many countries have followed this recommendation. In Sweden so far the recommendation is vaccination of identified risk groups for hepatitis B. Health care workers who are at risk of having blood contact in their work is one such risk group.In a large study on health care workers who were intradermally vaccinated with the hepatitis B vaccine, 960/1406 (68.3%) developed protective levels of antibodies to HBsAg (anti-HBs; defined as >10 mIU/mL) after three doses. After administering of an additional fourth dose to non-responders the response rate was 1187/1335 (88.9%). Risk factors for non-response were smoking and age above 40 years. Also, the vaccine response rates improved during the study and a risk of giving a too small dose with intradermal administration was also identified. This suggests that intradermal administration is dependent on well trained personnel.A genetic factor which has been proposed to be associated with a non-responder status to HBV vaccination is the HLA haplotype of the host. In a study in on 69 responders and 53 non-responders the haplotypes were therefore determined. It was found that [DQB1*0602; DQA1*0102; DR15] and [DQB1*0603; DQA1*0103; DRB1*1301] were more likely to be found in responders (p<0.025 and p<0.05 respectively). In non-responders the haplotype [DQB1*0604; DQA1*0102; DRB1*1302] was found more frequently (p<0.005). This study supports that the HLA class II of the host is involved in the ability to respond to the HBV vaccination.To further test the genetic link between the HLA of the host and a non-responder status, relatives to known intradermal non-responders with known haplotypes for DQA1, DQB1 and DRB1 were vaccinated in the same way, intradermally. The response rate in the relatives was 15/26 (58%) which is lower than expected suggesting a genetic influence on the vaccine response. In this study 5/6 with the haplotype [DQB1*0604; DQA1*0102; DRB1*1302] were non-responders which is in line with the previous data that this haplotype is correlated to hepatitis B vaccine non-response.Finally, to test a strategy by which we could induce an effective anti-HBs seroconversion in non-responders we revaccinated these with the combined hepatitis A and B vaccine intramuscularly at a double dose. Already after the first revaccination dose 26/44 (60%) responded with protective antibodies compared to 2/20 (10%) in a vaccine naïve reference group, suggesting an anamnestic response. After three doses 42/44 (95%) responded in the non-responder group and 20/20 (100%) in the reference group. All participants in the study responded to the hepatitis A antigen.In conclusion these studies show that intradermal vaccine administration can be used and is effective, and that the ability to respond is influenced by several, including genetic, factors. Importantly a non-responder status to hepatitis B vaccination is not absolute, a double dose of the combined HAV and HBV vaccine effectively overcomes this non-response in most individuals.
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| 3. |
- Vasconcelos, Nina-Maria, 1975-
(författare)
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Vaccine development strategies applied to the Plasmodium falciparum malaria antigen 332
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malaria is one of the major infectious diseases in the world with regard to mortality and morbidity, and the development of a vaccine against the malaria parasite Plasmodium falciparum is considered of high priority. The aim of the work presented in this thesis was to develop and characterize recombinant vaccine constructs based on the P. falciparum asexual blood-stage antigen Pf332. We have studied the humoral responses in mice elicited by various types of constructs, including naked DNA plasmids, naked mRNA, alphavirus, and peptides. Immunological memory was successfully induced against the repetitive EB200 fragment of Pf332, although the antibody titers were generally low and the highest titers were unexpectedly obtained with a conventional DNA plasmid. In another study, we also demonstrated the ability to circumvent genetically restricted immune responses in mice against two malaria epitopes, one of them derived from Pf332, by inclusion of universal T-cell epitopes into multiple antigen peptide constructs. However, the overall variability of the responses stressed the importance of including several epitopes in a future malaria vaccine. Further, the recent completion of sequencing of Pf332 enabled us to identify and characterize the immunogenic properties of a non-repeat fragment of the Pf332, termed C231. Our analyses of C231 showed that antibodies raised against the recombinant protein possess an in vitro parasite inhibitory capacity similar to that of antibodies against recombinant EB200. Furthermore, the recognition of C231 by antibodies in sera from individuals naturally primed to P. falciparum, correlated well with that previously observed for the corresponding sera and EB200. When analyzing the IgG subclass distribution of anti-C231 antibodies, we noted a bias towards IgG2 and IgG3 relative to IgG1, differing from the subclass profiles of IgG binding crude P. falciparum antigen, which were dominated by IgG1. Taken together, the work presented herein is likely to facilitate further studies on Pf332 as a potential target for protective immune responses, and amounts to a small step towards the realization of a malaria vaccine.
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