Browsing by master's degree program "Magisterprogrammet i farmaceutisk forskning, utveckling och säkerhet"

Cancer immunotherapy refers to therapy strategies that utilise the mechanisms of the immune system to treat cancer patients. The benefits of the approach include the possibility for specific targeting and utilisation of the host immune system. The treatment methods include cancer vaccines, oncolytic viruses (OVs), cell-based immunotherapies and antibodies. The interplay between the cancer and the immune system has been observed crucial for the progress of the cancer and the success of immunotherapies. An immune inflamed tumour microenvironment has been observed beneficial for the success of several therapy methods. Many immunotherapy methods rely on detecting tumour specific antigens that are used to guide the therapy agent to the target site. This strategy poses challenges when considering tumour immune evasion mechanisms, which can cause downregulation of target antigens, and heterogeneity of tumour cells and patients. OVs have the advantage of not requiring predetermined target structures to exert their effect to the tumour cells. They cause direct tumour cell lysis and induce immune responses, and may be modified to express additional genes, including immunostimulatory agents. However, virus-related immunosuppressive mechanisms and a rapid viral clearance may limit their effects. A Western Reserve (WR) Vaccinia virus (VACV) is a highly oncolytic virus strain but the virus has been observed to suppress the function of the cyclic guanosine monophosphate adenosine monophosphate synthase – stimulator of interferon genes (cGAS STING) innate immune pathway which has been shown to have a significant role in anti-tumour immune responses. The aim of this study was to create a WR VACV encoding a dominantly active (D A) STING and to determine whether the virus is capable of activating the cGAS STING pathway. The effects were compared to a corresponding virus vvdd tdTomato that does not have the STING encoding gene. The pathogenicity of viruses was controlled by a double deletion of the thymidine kinase and vaccinia growth factor genes which restricts the virus replication to tumour cells. Transgene fragments were cloned from template plasmids by polymerase chain reactions (PCRs) and joined together in a Gibson Assembly (GA) reaction to form a STING-P2A-eGFP gene insert. The insert was attached to a shuttle vector pSC65-tdTomato by restriction enzyme digestion, ligation and transformation in Escherichia coli. The correct transgene plasmid construct was verified by Sanger sequencing and PCRs. The transgene was inserted to a modified WR VACV vvdd-tdTomato-hDAI by a homologous recombination. The newly created VVdd STING-P2A-eGFP virus was purified by plaque purification. The STING protein expression was studied by an immunocytochemistry (ICC) assay. The immune signalling pathway activation was examined by testing nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation in RAW-Blue cells and dendritic cell activation and maturation in JAWS II cells. The cell viability after iinfection was studied with four cell lines; A549, B16-F10, HEK293 and MB49. The D-A STING expressing virus was produced successfully. The ICC experiment verified the capability of the VVdd STING-P2A eGFP to produce the STING protein in the infected cells. The preliminary findings indicate that the VVdd STING-P2A-eGFP virus activates the NF-κB signalling in the RAW-Blue cells and that the activation is dependent on the STING expression. The activation level is relative to the infection concentration at MOI range 0,001 to 0,1. The findings suggest that the VVdd-STING-eGFP virus can induce innate immune signalling via the STING pathway. The reference virus did not activate the signalling. The in vitro experiments also indicated that the STING virus may induce DC activation and maturation. We observed a trend of CD86 and CD40 expression upregulation on the JAWS II DCs. The effects to the cell viability were inconclusive. More studies should be conducted to verify the results. The effects of the virus should be studied in more advanced cancer models that take into account the complexity of the immune system. These preliminary results indicate the that the VVdd-STING-P2A-eGFP virus could stimulate the immune signalling through the STING pathway.

Antibiotic-resistant bacterial infections are a silently spreading pandemic that endangers public health and the healthcare system globally. Common infections may become more life-threatening, and hospital-acquired multi-drug-resistant infections soon compromise all medical procedures, such as surgeries and chemotherapy treatments. Two major players affecting the effectiveness of antimicrobial therapy against polymicrobial infections are interactions between bacterial species and biofilm formation. Bioiflm-embedded cells are protected from various threats, such as the host immune system and antibiotic interventions in the commensal polymicrobial community of increased virulence. Given the increasingly limited options of antibiotics against biofilm-associated antimicrobial-resistant infections, novel therapeutic strategies are needed. Phage therapy has regained interest as a promising strategy for treating antibiotic-resistant bacterial infections and limiting the evolution of resistance. In particular, phage-antibiotic combination therapy has been shown to be more efficient in treating pathogenic bacteria than using either one alone. In this study, I aimed to find a phage-antibiotic combination therapy against the formation of single and dual-species biofilm of S. aureus and P. aeruginosa and demonstrate the therapeutic potential of phages in combination with antibiotics by using a simple but clinically relevant in vitro biofilm model that supports the concomitant growth of P. aeruginosa and S. aureus. I found out that using phage Stab21 with ciprofloxacin or vancomycin alone or in combination was more effective in preventing the biofilm formation of S. aureus than using phage or antibiotic therapy alone. This was observed in the single-species biofilm of S. aureus and the dual-species biofilm in coculture with P. aeruginosa. Even though Stab21 alone could not infect S. aureus in liquid culture, adding ciprofloxacin or vancomycin at sublethal concentrations increased phage production.

Antimicrobial resistance (AMR) is a growing global health concern, and the development of new antibacterial agents is crucial to addressing this issue. Commercial antibiotics are not as effective as they used to be to combat infections. Previous studies have demonstrated the promising antimicrobial activity of etrasimod and one of its derivatives, compound 24f, against Gram-positive species. Therefore, as part of this study, we modified the carboxylic acid functional group to produce new derivatives. We synthesized derivatives of etrasimod and 24f, in order to generate a variety of compounds for evaluation of their antimicrobial effectiveness. Furthermore, the study evaluates the compounds' in vitro antibacterial activity and in vivo efficacy using Caenorhabditis elegans worms as an infection model. C. elegans is a widely used model organism in biological research, and it is particularly useful for studying host-pathogen interactions and drug efficacy. In addition, the cytotoxicity on mammalian cells (HeLa) was determined. Compound 18 showed the lowest cytotoxicity level (CC50 = 75.71±14.4 µM) of tested compounds. The antibacterial activity of new etrasimod derivatives was tested against Gram-positive (Staphylococcus aureus, including methicillin-resistant strains (MRSA)) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). The tested compounds showed activity against Gram-positive bacteria but not against any of the Gram-negative strains. Compounds 9 and 18 showed to be the most active compounds, having a minimum inhibitory concentration of 5–6 and 8–10 μM, respectively. Moreover, both compounds showed promising activity in vivo, being able to significantly reduce the bacterial load in infected worms and improve their survival rates in survival experiments. The study provides insights into the development and assessment of potential antibacterial agents, addressing the contemporary challenge of AMR. The study's findings suggest that compounds 9 and 18 could be potential candidates for further development as novel antimicrobial agents.

In recent years, animals have been recognized as promising next-generation protein production systems. Animal transgenesis has been achieved primarily in insect cells infected by recombinant baculoviruses. Baculovirus Expression Vector Systems (BEVS) transform the DH10Bac strain of Escherichia coli with the shuttle vector to produce recombinant baculovirus carrying the cargo of interest. The cargo includes at least one promoter driving the expression of at least one protein. PGK is a strong promoter that is naturally active in almost all species where it has been tested, including invertebrates like Drosophila. The PiggyBac transposon-based system is a known strategy for genome integration of foreign genes to create transgenic animals. Nevertheless, nobody has used baculoviruses to deliver genes and produce proteins in earthworms nor to create transgenic earthworms. There is also no information on the sequence of any endogenous E. fetida (earthworm) promoter yet. This project aimed to pilot a novel gene delivery method by creating baculoviruses through the BEVS, carrying the PGK promoter and the GFP reporter gene, and to assess the promoter activity in both Sf9 insect cells and E. fetida through evaluation of GFP fluorescence. Another target was to test the fluorescence after the addition to the baculovirus of the PiggyBac-based inverted terminal repeats (ITRs), flanking the PGK-GFP transcriptional unit. The secondary objective was to develop a non-lethal method for live worm imaging. Conventional restriction enzyme cloning was used to create the shuttle vectors, and restriction digest and Sanger Sequencing were used to identify the positive clones. The Bac-to-Bac BEVS was followed to create baculovirus particles carrying the cargo (PGK-GFP and PGK-GFP-ITR), infect Sf9 insect cells and monitor the PGK activity. Prior to in vitro transfection, the bacmid DNA was confirmed by PCR. These baculoviruses were also used to infect E. fetida and monitor the PGK activity in vivo. E. fetida autofluorescence was assessed before infection. PGK resulted in being much weaker in Sf9 than expected. The flanking of the transcriptional unit of GFP with the ITRs improved the GFP expression. 16% ethanol was shown to anaesthetize E. fetida for 10 to 15 minutes safely. Wild-type and starved E. fetida were shown to have very mild autofluorescence in their digestive system and setae. The coelomic fluid was shown to have strong autofluorescence. Thus, its excretion is crucial before imaging GFP. Likely, all the in vivo fluorescence after infection was due to the worm’s autofluorescence. Therefore, PGK and GFP were unlucky choices for E. fetida.