| 1. |
- Anderson, Jenna, et al.
(author)
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Molecular Signatures of a TLR4 Agonist-Adjuvanted HIV-1 Vaccine Candidate in Humans
- 2018
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In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9
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Journal article (peer-reviewed)abstract
- Systems biology approaches have recently provided new insights into the mechanisms of action of human vaccines and adjuvants. Here, we investigated early transcriptional signatures induced in whole blood of healthy subjects following vaccination with a recombinant HIV-1 envelope glycoprotein subunit CN54gp140 adjuvanted with the TLR4 agonist glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) and correlated signatures to CN54gp140-specific serum antibody responses. Fourteen healthy volunteers aged 18-45 years were immunized intramuscularly three times at 1-month intervals and whole blood samples were collected at baseline, 6 h, and 1, 3, and 7 days post first immunization. Subtle changes in the transcriptomic profiles were observed following immunization, ranging from over 300 differentially expressed genes (DEGs) at day 1 to nearly 100 DEGs at day 7 following immunization. Functional pathway analysis revealed blood transcription modules (BTMs) related to general cell cycle activation, and innate immune cell activation at early time points, as well as BTMs related to T cells and B cell activation at the later time points post-immunization. Diverse CN54gp140-specific serum antibody responses of the subjects enabled their categorization into high or low responders, at early (< 1 month) and late (up to 6 months) time points post vaccination. BTM analyses revealed repression of modules enriched in NK cells, and the mitochondrial electron chain, in individuals with high or sustained antigen-specific antibody responses. However, low responders showed an enhancement of BTMs associated with enrichment in myeloid cells and monocytes as well as integrin cell surface interactions. Flow cytometry analysis of peripheral blood mononuclear cells obtained from the subjects revealed an enhanced frequency of CD56(dim) NK cells in the majority of vaccines 14 days after vaccination as compared with the baseline. These results emphasize the utility of a systems biology approach to enhance our understanding on the mechanisms of action of TLR4 adjuvanted human vaccines.
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| 2. |
- Bahrami, F., et al.
(author)
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Blood transcriptional profiles distinguish different clinical stages of cutaneous leishmaniasis in humans
- 2022
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In: Molecular Immunology. - : Elsevier BV. - 0161-5890. ; 149, s. 165-173
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Journal article (peer-reviewed)abstract
- Cutaneous leishmaniasis (CL) is a neglected tropical disease with severe morbidity and socioeconomic sequelae. A better understanding of underlying immune mechanisms that lead to different clinical outcomes of CL could inform the rational design of intervention measures. While transcriptomic analyses of CL lesions were recently reported by us and others, there is a dearth of information on the expression of immune-related genes in the blood of CL patients. Herein, we investigated immune-related gene expression in whole blood samples collected from individuals with different clinical stages of CL along with healthy volunteers in an endemic CL region where Leishmania (L.) tropica is prevalent. Study participants were categorized into asymptomatic (LST+) and healthy uninfected (LST-) groups based on their leishmanin skin test (LST). Whole blood PAXgene samples were collected from volunteers, who had healed CL lesions, and patients with active L. tropica cutaneous lesions. Quality RNA extracted from 57 blood samples were subjected to Dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) assay for profiling 144 immune-related genes. Results show significant changes in the expression of genes involved in interferon signaling pathway in the blood of active CL patients, asymptomatics and healed individuals. Nonetheless, distinct profiles for several immune-related genes were identified in the healed, the asymptomatic, and the CL patients compared to the healthy controls. Among others, IFI16 and CCL11 were found as immune transcript signatures for the healed and the asymptomatic individuals, respectively. These results warrant further exploration to pinpoint novel blood biomarkers for different clinical stages of CL.
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| 3. |
- Del Campo, Judith, et al.
(author)
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Intranasal immunization with a proteoliposome-derived cochleate containing recombinant gD protein confers protective immunity against genital herpes in mice.
- 2010
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In: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 28:5, s. 1193-200
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Journal article (peer-reviewed)abstract
- The purpose of this study was to investigate the potential of intranasal (IN) immunization with Neisseria meningitides B proteoliposome (AFPL1) and AFPL1-derived cochleate (AFCo1), containing glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) for induction of protective immunity against genital herpes infection in mice. We could show that IN immunization with both AFPL1 and AFCo1 containing gD induced gD-specific IgG antibody and lymphoproliferative responses. However, IFN-gamma response could only be detected in CD4(+) splenic cells and genital lymph node cells of the AFCo1gD immunized mice upon recall antigen stimulation in vitro. Importantly, IN immunization with AFCo1gD could elicit a complete protection against an otherwise lethal vaginal challenge with HSV-2, while the AFPL1gD immunized mice were only partially protected. Further, we could show that the IFN-gamma response and protective immunity observed after IN immunization with AFCo1gD are mediated via the adaptor molecule myeloid differentiation factor 88. These data may have implications for the development of a mucosal vaccine against genital herpes.
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| 4. |
- Hammarström, Per, 1972-, et al.
(author)
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Protein substrate binding induces conformational changes in the chaperonin GroEL : A suggested mechanism for unfoldase activity
- 2000
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:30, s. 22832-22838
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Journal article (peer-reviewed)abstract
- Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxy1-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.
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| 5. |
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| 6. |
- Masoudzadeh, Nasrin, et al.
(author)
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Molecular signatures of anthroponotic cutaneous leishmaniasis in the lesions of patients infected with Leishmania tropica.
- 2020
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- Anthroponotic cutaneous leishmaniasis (CL) caused by Leishmania tropica (L. tropica) represents a public health challenge in several resource poor settings. We herein employed a systems analysis approach to study molecular signatures of CL caused by L. tropica in the skin lesions of ulcerative CL (UCL) and non-ulcerative CL (NUCL) patients. Results from RNA-seq analysis determined shared and unique functional transcriptional pathways in the lesions of the UCL and NUCL patients. Several transcriptional pathways involved in inflammatory response were positively enriched in the CL lesions. A multiplexed inflammatory protein analysis showed differential profiles of inflammatory cytokines and chemokines in the UCL and NUCL lesions. Transcriptional pathways for Fcγ receptor dependent phagocytosis were among shared enriched pathways. Using L. tropica specific antibody (Ab)-mediated phagocytosis assays, we could substantiate Ab-dependent cellular phagocytosis (ADCP) and Ab-dependent neutrophil phagocytosis (ADNP) activities in the lesions of the UCL and NUCL patients, which correlated with L. tropica specific IgG Abs. Interestingly, a negative correlation was observed between parasite load and L. tropica specific IgG/ADCP/ADNP in the skin lesions of CL patients. These results enhance our understanding of human skin response to CL caused by L. tropica.
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| 7. |
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| 8. |
- Persson, Malin, 1968-
(author)
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The Interaction Between the Chaperonin GroEL and Human Carbonic Anhydrase II : Protein Folding and Stability Studies
- 1998
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Doctoral thesis (other academic/artistic)abstract
- The presence of GroEL/ES during the refolding of human carbonic anhydrase II was found to increase the yield of active enzyme from approx. 70 to approx. 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonican hydrase, in which N-terrninal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.The chaperonin GroEL is a heat shock protein, i.e. it protects other proteins when they are exposed to elevated temperatures. To investigate if GroEL protects HCA II from thermal denaturation we studied the interaction between GroEL and HCA II at different temperatures. The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35°C. At temperatures above 35°C, the enzymatic activity is not stable, but decreases overtime. If GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50°C. However, unlike the spontaneous reactivation, the enzymatic activity is stable with time in the presence of GroEL. In the presence of the chaperonin, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, praline isomerization is part of the rate limiting step of refolding even in the presence of GroEL, and it is noteworthy that prolyl isomerase will influence the refolding of HCA II inthe presence of GroEL.The kinetics of the refolding of HCA II, at different temperatures, together with GroEL, has also been studied. The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (Ea) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively). This is the first indications that point to an active role for GroEL in the protein-folding process. Hence, GroEL does not only protect HCA II during refolding, it also assists the protein in the refolding reaction by providing a folding route with a flatter energy landscape than the spontaneous reaction.To further investigate the interaction between GroEL and HCA II, we have used electron paramagnetic resonance (EPR) to study HCA II cysteine mutants, spin-labeled at specific positions of the protein molecule. From the change of the mobility at temperatures between 20°C and 50°C of the spin-label for various mutants, with and without the presence of GroEL, the following general observations were made; i) HCA II appears to be unfolded to a degree similar to that of a GuHCl-induced molten-globule intermediate of the enzyme for the initial interaction with GroEL; ii) the degree of binding to GroEL is dependent on the stability of the HCA II variant; iii) GroEL efficiently protects HCA II from irreversible aggregation at higher temperatures; iv) the GroEL interaction leads to higher flexibility of the rigid and compact hydrophobic core, which is likely to facilitate rearrangements of misfolded structure; v) protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements.
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| 9. |
- Vianello, Eleonora, et al.
(author)
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Global blood miRNA profiling unravels early signatures of immunogenicity of Ebola vaccine rVSVΔG-ZEBOV-GP
- 2023
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In: iScience. - 2589-0042. ; 26:12
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Journal article (peer-reviewed)abstract
- The vectored Ebola vaccine rVSVΔG-ZEBOV-GP elicits protection against Ebola Virus Disease (EVD). In a study of forty-eight healthy adult volunteers who received either the rVSVΔG-ZEBOV-GP vaccine or placebo, we profiled intracellular microRNAs (miRNAs) from whole blood cells (WB) and circulating miRNAs from serum-derived extracellular vesicles (EV) at baseline and longitudinally following vaccination. Further, we identified early miRNA signatures associated with ZEBOV-specific IgG antibody responses at baseline and up to one year post-vaccination, and pinpointed target mRNA transcripts and pathways correlated to miRNAs whose expression was altered after vaccination by using systems biology approaches. Several miRNAs were differentially expressed (DE) and miRNA signatures predicted high or low IgG ZEBOV-specific antibody levels with high classification performance. The top miRNA discriminators were WB-miR-6810, EV-miR-7151-3p, and EV-miR-4426. An eight-miRNA antibody predictive signature was associated with immune-related target mRNAs and pathways. These findings provide valuable insights into early blood biomarkers associated with rVSVΔG-ZEBOV-GP vaccine-induced IgG antibody responses.
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| 10. |
- Vono, M., et al.
(author)
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C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates
- 2023
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In: FRONTIERS IN IMMUNOLOGY. - : Frontiers Media SA. - 1664-3224. ; 14
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Journal article (peer-reviewed)abstract
- Introduction: C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults. Methods: Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF (R) 01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses. Results: On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01-induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)specific B cells to form HEL+ GC B cells in neonatal mice upon vaccination with HEL-OVA. Discussion: Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.
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