| 1. |
- Lauren, Ida, et al.
(författare)
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Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology
- 2022
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Ingår i: Immunity, Inflammation and Disease. - : John Wiley & Sons. - 2050-4527. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. Methods: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFN gamma) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. Results: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFN gamma and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFN gamma in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. Conclusion: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.
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| 2. |
- Dillner, Joakim, et al.
(författare)
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Antibodies to SARS-CoV-2 and risk of past or future sick leave
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The extent that antibodies to SARS-CoV-2 may protect against future virus-associated disease is unknown. We invited all employees (n=15,300) at work at the Karolinska University Hospital, Stockholm, Sweden to participate in a study examining SARS-Cov-2 antibodies in relation to registered sick leave. For consenting 12,928 healthy hospital employees antibodies to SARS-CoV-2 could be determined and compared to participant sick leave records. Subjects with viral serum antibodies were not at excess risk for future sick leave (adjusted odds ratio (OR) controlling for age and sex: 0.85 [95% confidence interval (CI) (0.85 (0.43-1.68)]. By contrast, subjects with antibodies had an excess risk for sick leave in the weeks prior to testing [adjusted OR in multivariate analysis: 3.34 (2.98-3.74)]. Thus, presence of viral antibodies marks past disease and protection against excess risk of future disease. Knowledge of whether exposed subjects have had disease in the past or are at risk for future disease is essential for planning of control measures.Trial registration: First registered on 02/06/20, ClinicalTrials.gov NCT04411576.
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| 3. |
- Havervall, Sebastian, et al.
(författare)
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Robust humoral and cellular immune responses and low risk for reinfection at least 8 months following asymptomatic to mild COVID-19
- 2022
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Ingår i: Journal of Internal Medicine. - : John Wiley & Sons. - 0954-6820 .- 1365-2796. ; 291:1, s. 72-80
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Tidskriftsartikel (refereegranskat)abstract
- Background: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts.Methods: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points.Results: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%).Conclusions: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.
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| 4. |
- Havervall, Sebastian, et al.
(författare)
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SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection
- 2022
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 17:1, s. e0262169-e0262169
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Tidskriftsartikel (refereegranskat)abstract
- Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10−23 and 2*10−13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys
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| 5. |
- Hedhammar, My, Professor, 1975-, et al.
(författare)
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Zbasic : A Purification Tag for Selective Ion-Exchange Recovery
- 2021
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Ingår i: Protein Downstream Processing. - New York, NY : Humana Press. ; , s. 149-158
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Bokkapitel (refereegranskat)abstract
- A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein. Herein, protocols for purification of soluble Zbasic-tagged fusion proteins, as well as for integrated purification and solid-phase refolding of insoluble fusion proteins, are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.
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| 6. |
- Hober, Sophia, Professor, 1965-, et al.
(författare)
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Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay
- 2021
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Ingår i: Clinical & Translational Immunology. - : Wiley. - 2050-0068. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Objective. The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. Methods. More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. Results. Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. Conclusion. These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.
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| 7. |
- Jönsson, Malin
(författare)
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Engineering alternative scaffold proteins for conditional targeting
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Engineering naturally occurring proteins enables us to customize affinity domains according to our specific needs, tailoring them to become an important tool in a wide array of applications limited merely by our creativity. One of nature’s ways to regulate protein activity is by creating a functional change through alteration of a protein’s tertiary structure upon interaction with metal ions. Inspired by this elegant solution, this thesis has focused on engineering calcium-regulated affinity proteins using two different strategies and protein scaffolds.The first strategy revolved around designing and selecting a calcium- binding motif that can render the inherent target affinity of a naturally occurring protein domain to be turned on or off depending on whether calcium is present or not. The subject of this part of the thesis was one of the immunoglobulin-binding domains derived from Streptococcal Protein G. A library of various loops with prerequisites for attracting calcium was inserted between the IgG-binding surfaces of the domain prior to performing cell display selections aimed for rendering the inherent target interaction dependent on the presence of calcium. Successful selections resulted in a calcium-dependent version of the IgG-binding protein and its structure could be solved using NMR. A deeper investigation of the incorporated structural calcium-dependency could explain the underlying mechanisms giving rise to the functional on-and-off switch in target affinity and show how it derived from the evolutionary selection pressures applied.The second strategy included the creation of a combinatorial library based on a calcium-dependent protein scaffold, derived from Staphylococcal Protein A, for development of small calcium-regulated affinity – CaRA –imolecules with novel target specificities. Mimicking the multifaceted usefulness of naturally occurring metalloproteins, this second part of the thesis aimed at performing phage display selection campaigns towards a diverse set of targets relevant for various applications from bioprocessing (e.g. scFv) to biological therapies (e.g. TNFa, IL-23, EGFR). When evaluating the binding properties in the presence and absence of calcium, all discovered CaRA variants display calcium-dependent binding and target affinities in the nanomolar range.Engineering conditional binding can enhance the potential of next generation therapies in several ways. When used as calcium-dependent affinity ligands, it enables mild purification at neutral pH of therapeutic antibodies and antibody fragments that was previously limited by harsh acidic elution conditions. Reducing the risk of aggregated product by eluting at neutral pH would result in improved safety as well as the possibility to manufacture a greater repertoire of antibody formats. Furthermore, the conferred calcium- dependency of the CaRA scaffold can be used in a therapeutic approach envisioned to result in increased tissue penetration due to its small size and improved intracellular delivery by taking advantage of the existing calcium- gradient across the endosomal membrane of cells. This could lead to higher therapeutic efficacy by enabling lower doses or dosing frequency, further advancing a more patient-friendly future.
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| 8. |
- Mravinacová, Sára, et al.
(författare)
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A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies
- 2022
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 66, s. 46-52
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Tidskriftsartikel (refereegranskat)abstract
- Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.
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| 9. |
- Scheffel, Julia
(författare)
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Calcium-dependent Affinity Domains for the Purification of Antibodies and Antibody Fragments
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibodies are essential proteins in both our bodies and biotechnological research, and hold outstanding therapeutic value. The market for antibody-based therapeutics has grown exponentially during the last decades, owing to several advantages over small molecule drugs, such as fewer undesirable side effects associated with a higher target specificity. To keep up with the increasing amounts of antibodies that are on demand, emphasis has been on the optimization of upstream processes for antibody production while the advances in downstream processing and the purification of antibodies have been limited. In the downstream process, the gold standard for the primary capture step is Protein A affinity chromatography. However, elution of the antibodies from the Protein A ligand is accomplished at a low pH, which can lead to antibody aggregation and impaired biological activity. The purification procedure therefore hinders the development of new antibodies that are acid-sensitive, despite promising therapeutic potential, and may pose a threat to the increasingly popular bispecific antibodies that tend to be more aggregation prone. Further, acidic elution conditions may be an even bigger concern in the purification of antibody fragments, which also represent promising therapeutic candidates, providing several advantages over full-length antibodies in certain applications.The work in this thesis aimed to enable the purification of a more diverse group of antibodies and antibody fragments, regardless of their stability in a highly acidic environment. Efforts were also made to reduce the high antibody manufacturing costs to make these antibody therapeutics more easily accessible to patients. In order to elute the antibodies in the Protein A capture step under milder conditions, the protein ligand ZCa was developed. ZCa was isolated from a phage display library based on a Protein A domain with a grafted calcium-binding loop, and permits the calcium-dependent elution of antibodies at close to neutral pH by adding sodium chloride. The domain provides the foundation for this thesis, and was extensively optimized to achieve a high-capacity resin and an inexpensive purification strategy, yielding exceptional recoveries of pure antibody. Most importantly, ZCa was able to entirely prevent the formation of aggregates of an antibody through mild elution. Moreover, the optimized ZCa resin was applied in an integrated continuous biomanufacturing process, designed to entirely avoid the use of low pH. The implementation of the process at pilot scale for 17 days demonstrated the robustness of the novel resin along with many other promising process attributes. High productivity and yields were obtained in addition to negligible aggregate formation and low levels of residual DNA and host cell proteins, comparable to established processes.Finally, this thesis presents a combinatorial library for calcium-regulated affinity constructed from ZCa, from which numerous binders with novel target specificities were isolated. The calcium-dependent binders to single chain variable fragments (scFvs) could be used to selectively capture and elute scFv at neutral pH. Binding analysis and optimization efforts indicated potential as a platform approach for the mild and efficient purification of different scFvs.In conclusion, the purification strategies proposed in this thesis considerably improve the purification of antibodies and scFvs, and may encourage the future innovation of a wider range of antibody-based therapeutics. The continuous process supports the applicability of ZCa in a state-of-the-art commercial manufacturing process, and contributes to the more efficient manufacturing of antibodies, which can make them more affordable and accessible to the patients in need.
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| 10. |
- von Witting, Emma
(författare)
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The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Molecular recognition, or the specific interactions between a protein and its ligand, is central to biology and a key factor for many different clinical and technical applications. Despite antibodies being only one of many different affinity proteins, it has by far been the most successful. However, their large size and complex structure can be limiting in terms of cost and stability. Furthermore, their effector functions can sometimes be undesired or even detrimental. Over the past decades, many alternative affinity proteins have emerged to overcome some of these limitations.The Albumin Binding Domain (ABD), originally present on the surface of certain bacterial cells, has previously been subjected to combinatorial protein engineering for the generation of ADAPTs (ABD Derived Affinity ProTeins) that bind to different targets. One of these, the ADAPT6, targets HER2 and has shown great promise as a tracer for radionuclide molecular imaging for diagnosis and stratification of HER2 positive patients. The work in this thesis has aimed to optimise the ADAPT6 tracer further and also describes the first-inhuman clinical trial for imaging of HER2-overexpressing breast cancer. The results establish that ADAPT6 is safe and well-tolerated by patients and able to detect primary tumours as well as metastases with very high contrast already 2 hours after injection. However, the high kidney uptake associated with its fast blood clearance prevents further use of ADAPT6 also in a therapeutic setting. By engineering the ADAPT6 to prolong its circulatory half-life and reduce the kidney uptake, this thesis has also aimed to explore the therapeutic potential of this molecule. As a first step towards this goal, the ADAPT6 was genetically fused to an ABD to allow for binding to a patient’s own serum albumin and hence avoid the same extent of renal filtration. Indeed, when evaluated in mice, fusion to ABD increased the retention in circulation by more than 200-fold and exhibited a dramatically decreased renal activity. Treatment of tumour-bearing mice with the ABD-fused ADAPT6 conjugated to a cytotoxic radionuclide significantly prolonged survival by more than two-fold and was not associated with any observable toxicity. Finally, this thesis also describes a novel combinatorial library from which several bispecific ADAPTs have been identified, binding to both albumin and other clinically relevant targets simultaneously. This miniature bispecific scaffold offers an opportunity to combine the benefits associated with small size such as good tissue extravasation and alternative administration routes while still maintaining a sufficient in vivo half-life.
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| 11. |
- Wisniewski, Andreas, 1991-
(författare)
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Bifunctional ADAPTs: Opportunity for serological Half-life extension and Targeted therapy
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Small engineered scaffold proteins (ESPs) gain more and more popularity as biological drugs, due to their specificity and applicability in diagnostics and therapy. Thanks to their high stability, low immunogenicity and low production cost, they present themselves as a promising alternative to the market-leading antibodies. However, their relatively small size poses the risk of fast blood clearance, a circumstance advantageous for imaging purposes but a disadvantage in a therapeutic setting.This thesis has focused on introducing bifunctionality, the ability of the same engineered scaffold protein to exert more than one function, by applying different engineering approaches. Therefore, a new combinatorial protein library based on ABD-derived affinity proteins (ADAPTs) was generated, originating from a bacterial albumin-binding domain. From this library, it was possible to achieve protein modules with the ability to simultaneously bind to its intended target as well as to human serum albumin (HSA), a feature that has been shown to increase the binder’s half-life in the body. Specific binding modules were achieved by performing phage display selections towards the targets Tumor necrosis factor alpha (TNF⍺) and Interleukin-17c (IL-17c), both proinflammatory cytokines involved in many different inflammatory diseases and therefore interesting targets for therapeutic applications. The selection output was analyzed through sequencing and promising candidates were cloned and produced in Escherichia coli (E. coli), followed by a detailed characterization of each candidate including target binding, stability and their oligomeric state using methods like Surface Plasmon Resonance (SPR), Circular Dichroism (CD) and Size Exclusion Chromatography (SEC). It was possible to generate binders that passed all characterization criteria, most importantly showing simultaneous bispecificity to either TNF⍺ or IL-17c in combination with albumin. Each binder was then examined for their usefulness as a real therapeutic by successfully evaluating its ability to block the interaction of the cytokine and its specific receptor in vitro. These newly developed protein binders, showing high affinity towards their targets as well as keeping their initial binding to HSA, present another possibility to combine the advantages of small engineered scaffold proteins with those of typical larger proteins, allowing for more convenient production in bacteria leading to lower production costs and making them ideal candidates for future therapeutics.Furthermore, a previously developed ADAPT targeting the human epidermal growth factor receptor 2 (HER2) was genetically fused to an improved Horseradish Peroxidase (HRP) variant, thereby combining the idea of tumor-targeted therapy through the ADAPT with the utilization of HRP to enzymatically catalyze the prodrug IAA into its active form. After proving these new fusion proteins have similar binding kinetics to the target, as well as comparable enzymatic activities, as their free counterpart, the cytotoxic effects were put to the test in vitro. Hereby, the variants showed to benefit immensely through the addition of an ADAPT by being selectively effective only on HER2-positive cells. The evident advantage of these fusion proteins and their competency to be functionally produced in E. coli as well as the possibility to avoid an additional step of conjugation or coupling of affinity proteins to cytotoxic payloads, makes this approach a promising alternative for current procedures and another reason why ESPs are on the rise.
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| 12. |
- Abouzayed, Ayman, et al.
(författare)
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The GRPR Antagonist [Tc-99m]Tc-maSSS-PEG(2)-RM26 towards Phase I Clinical Trial : Kit Preparation, Characterization and Toxicity
- 2023
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Ingår i: Diagnostics. - : MDPI AG. - 2075-4418. ; 13:9, s. 1611-
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Tidskriftsartikel (refereegranskat)abstract
- Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [Tc-99m]Tc-maSSS-PEG2-RM26 (based on [D-Phe(6), Sta(13), Leu(14)-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 degrees C was monitored for 18 months. The biological properties of [Tc-99m]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG(2)-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16-24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.
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| 13. |
- Alkharaan, Hassan, et al.
(författare)
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Persisting Salivary IgG Against SARS-CoV-2 at 9 Months After Mild COVID-19 : A Complementary Approach to Population Surveys
- 2021
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 224:3, s. 407-414
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Tidskriftsartikel (refereegranskat)abstract
- Background. Declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short lived although long-term studies are lacking. Methods. Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n = 74, cohort 1), undiagnosed individuals with self-reported questionnaires (n = 147, cohort 2), and individuals sampled prepandemic (n = 35, cohort 3). Results. Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months postrecovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in blood and saliva in most patients. Salivary IgA was rarely detected at this time point. In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms. Salivary IgG tolerated temperature and detergent pretreatments. Conclusions. Unlike SARS-CoV-2 salivary IgA that appeared short lived, specific saliva IgG appeared stable even after mild COVID-19, as for blood serology. This noninvasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.
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| 14. |
- Appelberg, S., et al.
(författare)
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A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells
- 2022
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 14:10
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Tidskriftsartikel (refereegranskat)abstract
- New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.
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| 15. |
- Asplund Högelin, K., et al.
(författare)
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B-cell repopulation dynamics and drug pharmacokinetics impact SARS-CoV-2 vaccine efficacy in anti-CD20-treated multiple sclerosis patients
- 2022
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Ingår i: European Journal of Neurology. - : Wiley. - 1351-5101 .- 1468-1331. ; 29:11, s. 3317-3328
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose: Recent findings document a blunted humoral response to SARS-CoV-2 vaccination in patients on anti-CD20 treatment. Although most patients develop a cellular response, it is still important to identify predictors of seroconversion to optimize vaccine responses. Methods: We determined antibody responses after SARS-CoV-2 vaccination in a real-world cohort of multiple sclerosis patients (n = 94) treated with anti-CD20, mainly rituximab, with variable treatment duration (median = 2.9, range = 0.4–9.6 years) and time from last anti-CD20 infusion to vaccination (median = 190, range = 60–1032 days). Results: We find that presence of B cells and/or rituximab in blood predict seroconversion better than time since last infusion. Using multiple logistic regression, presence of >0.5% B cells increased probability of seroconversion with an odds ratio (OR) of 5.0 (95% confidence interval [CI] = 1.0–28.1, p = 0.055), whereas the corresponding OR for ≥6 months since last infusion was 1.45 (95% CI = 0.20–10.15, p = 0.705). In contrast, detectable rituximab levels were negatively associated with seroconversion (OR = 0.05, 95% CI = 0.002–0.392, p = 0.012). Furthermore, naïve and memory IgG+ B cells correlated with antibody levels. Although retreatment with rituximab at 4 weeks or more after booster depleted spike-specific B cells, it did not noticeably affect the rate of decline in antibody titers. Interferon-γ and/or interleukin-13 T-cell responses to the spike S1 domain were observed in most patients, but with no correlation to spike antibody levels. Conclusions: These findings are relevant for providing individualized guidance to patients and planning of vaccination schemes, in turn optimizing benefit–risk with anti-CD20.
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| 16. |
- Bergman, Peter, et al.
(författare)
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Safety and efficacy of the mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of immunocompromised patients and healthy controls in a prospective open-label clinical trial
- 2021
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Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 74
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Tidskriftsartikel (refereegranskat)abstract
- Background: Patients with immunocompromised disorders have mainly been excluded from clinical trials of vaccination against COVID-19. Thus, the aim of this prospective clinical trial was to investigate safety and efficacy of BNT162b2 mRNA vaccination in five selected groups of immunocompromised patients and healthy controls.Methods: 539 study subjects (449 patients and 90 controls) were included. The patients had either primary (n=90), or secondary immunodeficiency disorders due to human immunodeficiency virus infection (n=90), allogeneic hematopoietic stem cell transplantation/CAR T cell therapy (n=90), solid organ transplantation (SOT) (n=89), or chronic lymphocytic leukemia (CLL) (n=90). The primary endpoint was seroconversion rate two weeks after the second dose. The secondary endpoints were safety and documented SARS-CoV-2 infection.Findings: Adverse events were generally mild, but one case of fatal suspected unexpected serious adverse reaction occurred. 72.2% of the immunocompromised patients seroconverted compared to 100% of the controls (p=0.004). Lowest seroconversion rates were found in the SOT (43.4%) and CLL (63.3%) patient groups with observed negative impact of treatment with mycophenolate mofetil and ibrutinib, respectively.Interpretation: The results showed that the mRNA BNT162b2 vaccine was safe in immunocompromised patients. Rate of seroconversion was substantially lower than in healthy controls, with a wide range of rates and antibody titres among predefined patient groups and subgroups. This clinical trial highlights the need for additional vaccine doses in certain immunocompromised patient groups to improve immunity.
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| 17. |
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| 18. |
- Blixt, L., et al.
(författare)
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Covid-19 in patients with chronic lymphocytic leukemia : clinical outcome and B- and T-cell immunity during 13 months in consecutive patients
- 2022
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Ingår i: Leukemia. - : Springer Nature. - 0887-6924 .- 1476-5551. ; 36:2, s. 476-481
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Tidskriftsartikel (refereegranskat)abstract
- We studied clinical and immunological outcome of Covid-19 in consecutive CLL patients from a well-defined area during month 1–13 of the pandemic. Sixty patients (median age 71 y, range 43–97) were identified. Median CIRS was eight (4–20). Patients had indolent CLL (n = 38), had completed (n = 12) or ongoing therapy (n = 10). Forty-six patients (77%) were hospitalized due to severe Covid-19 and 11 were admitted to ICU. Severe Covid-19 was equally distributed across subgroups irrespective of age, gender, BMI, CLL status except CIRS (p < 0.05). Fourteen patients (23%) died; age ≥75 y was the only significant risk factor (p < 0.05, multivariate analysis with limited power). Comparing month 1–6 vs 7–13 of the pandemic, deaths were numerically reduced from 32% to 18%, ICU admission from 37% to 15% whereas hospitalizations remained frequent (86% vs 71%). Seroconversion occurred in 33/40 patients (82%) and anti-SARS-CoV-2 antibodies were detectable at six and 12 months in 17/22 and 8/11 patients, respectively. Most (13/17) had neutralizing antibodies and 19/28 had antibodies in saliva. SARS-CoV-2-specific T-cells (ELISpot) were detected in 14/17 patients. Covid-19 continued to result in high admission even among consecutive and young early- stage CLL patients. A robust and durable B and/or T cell immunity was observed in most convalescents.
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| 19. |
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| 20. |
- Bragina, Olga, et al.
(författare)
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Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3, for Imaging of HER2-Positive Breast Cancer
- 2023
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 15:12
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary The receptor HER2 is overexpressed in some breast cancers. Tumours with a high HER2 expression can be successfully treated with the antibodies trastuzumab and pertuzumab. The radionuclide imaging of HER2 in disseminated cancer could help to select patients for treatment using these antibodies. Novel radiolabelled small-sized tracers, scaffold proteins, have shown excellent imaging properties in preclinical studies. The scaffold proteins [Tc-99m]Tc-ADAPT6 and DARPin [Tc-99m]Tc-(HE)(3)-G3 have been found to be safe in Phase I clinical trials. They showed promising results in the imaging of HER2. In this study, we compared the distribution of both tracers in the same patients with breast cancer to evaluate whether one of them has any decisive advantage. We found that both tracers provide an excellent visualization of tumours, but the accumulation of [Tc-99m]Tc-ADAPT6 in tumours is higher. The data from this study are essential for researchers developing imaging agents. Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [Tc-99m]Tc-ADAPT6 and DARPin [Tc-99m]Tc-(HE)(3)-G3 in breast cancer patients have demonstrated their safety and indicated their capability to discriminate between HER2-positive and HER2-negative tumours. The objective of this study was to compare the imaging of HER2-positive tumours in the same patients using [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3. Eleven treatment-naive female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study. Each patient was intravenously injected with [Tc-99m]Tc-ADAPT6, followed by an [Tc-99m]Tc-(HE)(3)-G3 injection 3-4 days later and chest SPECT/CT was performed. All primary tumours were clearly visualized using both tracers. The uptake of [Tc-99m]Tc-ADAPT6 in primary tumours (SUVmax = 4.7 & PLUSMN; 2.1) was significantly higher (p < 0.005) than the uptake of [Tc-99m]Tc-(HE)(3)-G3 (SUVmax = 3.5 & PLUSMN; 1.7). There was no significant difference in primary tumour-to-contralateral site values for [Tc-99m]Tc-ADAPT6 (15.2 & PLUSMN; 7.4) and [Tc-99m]Tc-(HE)(3)-G3 (19.6 & PLUSMN; 12.4). All known lymph node metastases were visualized using both tracers. The uptake of [Tc-99m]Tc-ADAPT6 in all extrahepatic soft tissue lesions was significantly (p < 0.0004) higher than the uptake of [Tc-99m]Tc-(HE)(3)-G3. In conclusion, [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3 are suitable for the visualization of HER2-positive breast cancer. At the selected time points, [Tc-99m]Tc-ADAPT6 has a significantly higher uptake in soft tissue lesions, which might be an advantage for the visualization of small metastases.
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| 21. |
- Bragina, Olga, et al.
(författare)
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Evaluation of Approaches for the Assessment of HER2 Expression in Breast Cancer by Radionuclide Imaging Using the Scaffold Protein [99mTc]Tc-ADAPT6
- 2024
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Due to its small size and high affinity binding, the engineered scaffold protein ADAPT6 is a promising targeting probe for radionuclide imaging of human epidermal growth factor receptor type 2 (HER2). In a Phase I clinical trial, [Tc-99m]Tc-ADAPT6 demonstrated safety, tolerability and capacity to visualize HER2 expression in primary breast cancer. In this study, we aimed to select the optimal parameters for distinguishing between breast cancers with high and low expression of HER2 using [Tc-99m]Tc-ADAPT6 in a planned Phase II study. HER2 expression was evaluated in primary tumours and metastatic axillary lymph nodes (mALNs). SPECT/CT imaging of twenty treatment-naive breast cancer patients was performed 2 h after injection of [Tc-99m]Tc-ADAPT6. The imaging data were compared with the data concerning HER2 expression obtained by immunohistochemical evaluation of samples obtained by core biopsy. Maximum Standard Uptake Values (SUVmax) afforded the best performance for both primary tumours and mALNs (areas under the receiver operating characteristic curve (ROC AUC) of 1.0 and 0.97, respectively). Lesion-to-spleen ratios provided somewhat lower performance. However, the ROC AUCs were still over 0.90 for both primary tumours and mALNs. Thus, lesion-to-spleen ratios should be further evaluated to find if these could be applied to imaging using stand-alone SPECT cameras that do not permit SUV calculations.
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| 22. |
- Bragina, Olga, et al.
(författare)
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Phase I study of 99mTc-ADAPT6, a scaffold protein-based probe for visualization of HER2 expression in breast cancer
- 2021
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 62:4, s. 493-499
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging of human epidermal growth factor (HER2) expression may be helpful to stratify breast and gastroesophageal cancer patients for HER2-targeting therapies. ADAPTs (albumin-binding domain derived affinity proteins) are a new type of small (46-59 amino acids) proteins useful as probes for molecular imaging. The aim of this first-in-human study was to evaluate biodistribution, dosimetry, and safety of the HER2-specific 99mTc-ADAPT6.METHODS: Twenty-nine patients with primary breast cancerwere included. In 22 patients with HER2-positive (n = 11) or HER2-negative (n = 11) histopathology an intravenous injection with 385±125 MBq 99mTc-ADAPT6 was performed, randomized to an injected protein mass of either 500 µg (n = 11) or 1000 µg (n = 11). Planar scintigraphy followed by SPECT imaging was performed after 2, 4, 6 and 24 h. An additional cohort (n = 7) was injected with 165±29 MBq (injected protein mass 250 µg) and imaging was performed after 2 h only.RESULTS: Injections of 99mTc-ADAPT6 at all injected mass levels were well tolerated and not associated with adverse effects. 99mTc-ADAPT6 cleared rapidly from blood and most other tissues. The normal organs with the highest accumulation were kidney, liver and lung. Effective doses were 0.009±0.002 and 0.010±0.003 mSv/MBq for injected protein masses of 500 and 1000 µg, respectively. Injection of 500 µg resulted in excellent discrimination between HER2-positive and HER2-negative tumors already 2 h after injection (tumor-to-contralateral breast ratio was 37±19 vs 5±2, p<0.01). The tumor-to-contralateral breast ratios for HER2-positive tumors were significantly (p<0.05) higher for injected mass of 500 µg than for both 250 and 1000 µg.CONCLUSION: Injections of 99mTc-ADAPT6 are safe and associated with low absorbed and effective doses. Protein dose of 500 µg is preferable for discrimination between tumors with high and low expression of HER2. Further studies are justified to evaluate if 99mTc-ADAPT6 can be used as an imaging probe for stratification of patients for HER2-targeting therapy in the areas where PET imaging is not readily available.
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| 23. |
- Dannemeyer, Melanie, et al.
(författare)
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Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors
- 2024
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 221
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Tidskriftsartikel (refereegranskat)abstract
- Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.
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| 24. |
- Ekblad, Caroline, et al.
(författare)
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Polypeptides based on a scaffold
- 2022
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Patent (populärvet., debatt m.m.)abstract
- The disclosure provides a population of polypeptide variants based on a common scaffold, each polypeptide in the population comprising the scaffold amino acid sequence Xsc1AELDXsc2Xsc3GVG AXXIKXIXsc4XA XXVEXVQXXK QXILAX. The disclosure also provides methods for selecting and identifying polypeptides from the population, as well as such polypeptides themselves.
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| 25. |
- Elfstrom, K. Miriam, et al.
(författare)
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Differences in risk for SARS-CoV-2 infection among healthcare workers
- 2021
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Ingår i: Preventive Medicine Reports. - : Elsevier BV. - 2211-3355. ; 24
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Tidskriftsartikel (refereegranskat)abstract
- Healthcare workers (HCWs) are a risk group for SARS-CoV-2 infection, but which healthcare work that conveys risk and to what extent such risk can be prevented is not clear. Starting on April 24th, 2020, all employees at work (n = 15,300) at the Karolinska University Hospital, Stockholm, Sweden were invited and 92% consented to participate in a SARS-CoV-2 cohort study. Complete SARS-CoV-2 serology was available for n = 12,928 employees and seroprevalences were analyzed by age, sex, profession, patient contact, and hospital department. Relative risks were estimated to examine the association between type of hospital department as a proxy for different working environment exposure and risk for seropositivity, adjusting for age, sex, sampling week, and profession. Wards that were primarily responsible for COVID-19 patients were at increased risk (adjusted OR 1.95 (95% CI 1.65-2.32) with the notable exception of the infectious diseases and intensive care units (adjusted OR 0.86 (95% CI 0.66-1.13)), that were not at increased risk despite being highly exposed. Several units with similar types of work varied greatly in seroprevalences. Among the professions examined, nurse assistants had the highest risk (adjusted OR 1.62 (95% CI 1.38-1.90)). Although healthcare workers, in particular nurse assistants, who attend to COVID-19 patients are a risk group for SARS-CoV-2 infection, several units caring for COVID-19 patients had no excess risk. Large variations in seroprevalences among similar units suggest that healthcare work-related risk of SARS-CoV-2 infection may be preventable.
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| 26. |
- Garousi, Javad, et al.
(författare)
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Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts : Efficacy and Selection of Companion Imaging Counterpart
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 14:8
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPT(Neg)-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as Tc-99m(CO)(3)-ADAPT6 or the affibody molecule Tc-99m-Z(HER2:41071), are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1-based therapy.
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| 27. |
- Garousi, Javad, et al.
(författare)
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Radionuclide therapy using ABD-fused ADAPT scaffold protein : Proof of Principle
- 2021
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 266
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Tidskriftsartikel (refereegranskat)abstract
- Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.
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| 28. |
- Garousi, Javad, et al.
(författare)
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Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:11, s. 1847-
-
Tidskriftsartikel (refereegranskat)abstract
- Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with Tc-99m, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.
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| 29. |
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| 30. |
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| 31. |
- Havervall, Sebastian, et al.
(författare)
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Antibody responses after a single dose of ChAdOx1 nCoV-19 vaccine in healthcare workers previously infected with SARS-CoV-2
- 2021
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Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 70
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recent reports demonstrate robust serological responses to a single dose of messenger RNA (mRNA) vaccines in individuals previously infected with SARS-CoV-2. Data on immune responses following a single-dose adenovirus-vectored vaccine expressing the SARS-CoV-2 spike protein (ChAdOx1 nCoV-19) in individuals with previous SARS-CoV-2 infection are however limited, and current guidelines recommend a two-dose regimen regardless of preexisting immunity. Methods: We compared RBD-specific IgG and RBD-ACE2 blocking antibodies against SARS-CoV-2 wild type and variants of concern following two doses of the mRNA vaccine BNT162b2 in SARS-CoV-2 naive healthcare workers (n=65) and a single dose of the adenovector vaccine ChAdOx1 nCoV-19 in 82 healthcare workers more than (n=45) and less than (n=37) 11 months post mild SARS-CoV-2 infection at time of vaccination. Findings: The post-vaccine levels of RBD-specific IgG and neutralizing antibodies against the SARS-CoV-2 wild type and variants of concern including Delta lineage 1.617.2 were similar or higher in participants receiving a single dose of ChAdOx1 nCoV-19 vaccine post SARS-CoV-2 infection (both more than and less than 11 months post infection) compared to SARS-CoV-2 naive participants who received two doses of BNT162b2 vaccine. Interpretation: Our data support that a single dose ChAdOx1 nCoV-19 vaccine that is administered up to at least 11 months post SARS-CoV-2 infection serves as an effective immune booster. This provides a possible rationale for a single-dose vaccine regimen. (C) 2021 The Author(s). Published by Elsevier B.V.
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| 32. |
- Havervall, Sebastian, et al.
(författare)
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Impact of SARS-CoV-2 infection on vaccine-induced immune responses over time
- 2022
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Ingår i: Clinical & Translational Immunology (CTI). - : Wiley. - 2050-0068. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- Objective. To determine the long-term impact of prior SARS-CoV-2 infection on immune responses after COVID-19 vaccination. Methods. Using longitudinally collected blood samples from the COMMUNITY study, we determined binding (WHO BAU mL(-1)) and neutralising antibody titres against ten SARS-CoV-2 variants over 7 months following BNT162b2 in SARS-CoV-2-recovered (n = 118) and SARS-CoV-2-naive (n = 289) healthcare workers with confirmed prior SARS-CoV-2 infection. A smaller group with (n = 47) and without (n = 60) confirmed prior SARS-CoV-2 infection receiving ChAdOx1 nCoV-19 was followed for 3 months. SARS-CoV-2-specific memory T-cell responses were investigated in a subset of SARS-CoV-2-naive and SARS-CoV-2-recovered vaccinees. Results. Vaccination with both vaccine platforms resulted in substantially enhanced T-cell responses, anti-spike IgG responses and neutralising antibodies effective against ten SARS-CoV-2 variants in SARS-CoV-2-recovered participants as compared to SARS-CoV-2-naive participants. The enhanced immune responses sustained over 7 months following vaccination. Conclusion. These findings imply that prior SARS-CoV-2 infection should be taken into consideration when planning booster doses and design of current and future COVID-19 vaccine programmes.
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| 33. |
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| 34. |
- Healy, Katie, et al.
(författare)
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Salivary IgG to SARS-CoV-2 indicates seroconversion and correlates to serum neutralization in mRNA-vaccinated immunocompromised individuals
- 2022
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Ingår i: Med. - : Cell Press. - 2666-6359 .- 2666-6340. ; 3:2, s. 137-153.e3
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Tidskriftsartikel (refereegranskat)abstract
- Background: Immunocompromised individuals are highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Whether vaccine-induced immunity in these individuals involves oral cavity, a primary site of infection, is presently unknown.Methods: Immunocompromised patients (n = 404) and healthy controls (n = 82) participated in a prospective clinical trial (NCT04780659) encompassing two doses of the mRNA BNT162b2 vaccine. Primary immunodeficiency (PID), secondary immunodeficiencies caused by human immunodeficiency virus (HIV) infection, allogeneic hematopoietic stem cell transplantation (HSCT)/chimeric antigen receptor T cell therapy (CAR-T), solid organ transplantation (SOT), and chronic lymphocytic leukemia (CLL) patients were included. Salivary and serum immunoglobulin G (IgG) reactivities to SARS-CoV-2 spike were measured by multiplex bead-based assays and Elecsys anti-SARS-CoV-2 S assay.Findings: IgG responses to SARS-CoV-2 spike antigens in saliva in HIV and HSCT/CAR-T groups were comparable to those of healthy controls after vaccination. The PID, SOT, and CLL patients had weaker responses, influenced mainly by disease parameters or immunosuppressants. Salivary responses correlated remarkably well with specific IgG titers and the neutralizing capacity in serum. Receiver operating characteristic curve analysis for the predictive power of salivary IgG yielded area under the curve (AUC) = 0.95 and positive predictive value (PPV) = 90.7% for the entire cohort after vaccination.Conclusions: Saliva conveys vaccine responses induced by mRNA BNT162b2. The predictive power of salivary spike IgG makes it highly suitable for screening vulnerable groups for revaccination.
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| 35. |
- Hober, Sophia, Professor, 1965-, et al.
(författare)
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Mutant protein
- 2016
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Patent (populärvet., debatt m.m.)
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| 36. |
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| 37. |
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| 38. |
- Högelin, Klara Asplund, et al.
(författare)
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Development of humoral and cellular immunological memory against SARS-CoV-2 despite B cell depleting treatment in multiple sclerosis
- 2021
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 24:9
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Tidskriftsartikel (refereegranskat)abstract
- B cell depleting therapies (BCDTs) are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. Their possible impact on development of immunity to severe acute respiratory syndrome virus-2 (SARS-CoV-2) has raised concerns with the coronavirus disease 2019 (COVID-19) pandemic. We here evaluated the frequency of COVID-19-like symptoms and determined immunological responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs (COMBAT-MS; NCT03193866) and in eleven patients after vaccination, with a focus on BCDT. Almost all seropositive and 17.9% of seronegative patients on BCDT, enriched for a history of COVID-19-like symptoms, developed anti-SARS-CoV-2 T cell memory, and T cells displayed functional similarity to controls producing IFN-gamma and TNF. Following vaccination, vaccine-specific humoral memory was impaired, while all patients developed a specific T cell response. These results indicate that BCDTs do not abrogate SARS-CoV-2 cellular memory and provide a possible explanation as to why the majority of patients on BCDTs recover from COVID-19.
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| 39. |
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| 40. |
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| 41. |
- Jönsson, Malin, et al.
(författare)
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CaRA – A multi-purpose phage display library for selection of calcium-regulated affinity proteins
- 2022
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Ingår i: New Biotechnology. - : Elsevier B.V.. - 1871-6784 .- 1876-4347. ; 72, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.
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| 42. |
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| 43. |
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| 44. |
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| 45. |
- Kanje, Sara, 1986-, et al.
(författare)
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Engineering of Protein A for improved purification of antibodies and Fc-fused proteins
- 2020
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Ingår i: Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. - : Elsevier. ; , s. 35-54
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In industrial scale downstream processing of antibodies and Fc-fusion proteins, Protein A chromatography is the most commonly used purification method. As the treatments for many severe diseases shift toward biological drugs, in particular antibodies, the need for robust purification methods has increased. Since the 1970s, when Protein A debuted as an affinity ligand, the full protein and its domains have been extensively studied. Even if matrices based on unmodified Protein A historically met the process demands of industrial antibody manufacturing, there is room for improvement. The cleaning in place (CIP) methods required for safe reuse of the matrix in therapeutic protein purification pose a problem since the harsh conditions required can be harmful to the proteinaceous purification ligand. Further, the low pH used for elution can sometimes be detrimental to the target protein. Here, efforts to improve these features as well as capacity improvements for Protein A resins will be discussed.
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| 46. |
- Kharlamova, N., et al.
(författare)
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False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.
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| 47. |
- Klevebro, Susanna, et al.
(författare)
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Risk of SARS-CoV-2 exposure among hospital healthcare workers in relation to patient contact and type of care
- 2021
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Ingår i: Scandinavian Journal of Public Health. - : SAGE Publications. - 1403-4948 .- 1651-1905. ; 49:7, s. 707-712
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Tidskriftsartikel (refereegranskat)abstract
- We aimed to assess prevalence of IgG antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and factors associated with seropositivity in a large cohort of healthcare workers (HCWs). Methods: From 11 May until 11 June 2020, 3981 HCWs at a large Swedish emergency care hospital provided serum samples and questionnaire data. Presence of IgG antibodies to SARS-CoV-2 was measured as an indicator of SARS-CoV-2 exposure. Results: The total seroprevalence was 18% and increased during the study period. Among the seropositive HCWs, 11% had been entirely asymptomatic. Participants who worked with COVID-19 patients had higher odds for seropositivity: adjusted odds ratio 1.96 (95% confidence intervals 1.59–2.42). HCWs from three of the departments managing COVID-19 patients had significantly higher seroprevalences, whereas the prevalence among HCWs from the intensive care unit (also managing COVID-19 patients) was significantly lower. Conclusions: HCWs in contact with SARS-CoV-2 infected patients had a variable, but on average higher, likelihood for SARS-CoV-2 infections.
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| 48. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
- 2022
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 171-187
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Tidskriftsartikel (refereegranskat)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
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| 49. |
- Mangsbo, Sara, 1981-, et al.
(författare)
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An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses
- 2021
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:9
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Tidskriftsartikel (refereegranskat)abstract
- Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-gamma Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.
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