Hepatitis B core protein and bacteriophage AP205 and GA coat protein formed virus-like paricles for packaging and addressing

Elektroniskā versija nesatur pielikumusVīrusiem līdzīgās daļiņas (VLD) ir proteīnu struktūras, kas veidotas no viena vai vairāku veidu proteīniem, kas spējīgi pašsavākties kompleksās un augsti organizētās struktūrās. Simetriskās un multimērās struktūras dēļ VLD var tikt izmantotas divos veidos – kā perspektīvi svešu sekvenču nesēji un eksponētāji, un kā piegādes vektori ar iepakotu kravu tajos. Viena no daudzsološākajām VLD pielietojuma jomām ir vakcīnu izstrāde. Bakteriofāgi GA un AP205 ir vieni no mazākajiem un vienkāršākajiem vīrusiem, plaši izmantoti kā modeļobjekts molekulārajā bioloģijā. Apvalka proteīnu ekspresējot baktērijās vai raugos, veidojas VLD. Cita objekta – hepatīta B vīrusa (HBV) core proteīna gadījumā, tas kā rekombinants proteīns spēj pašsavākties vīrusiem-līdzīgās daļiņās. Darba mērķis bija pārbaudīt dažādas vīrusiem līdzīgo daļiņu pielietojuma iespējas, tāpēc tika izmantoti dažādi testa objekti. Pirmkārt, West-Nīlas vīrusa E proteīna DIII domēna (WNV-DIII) proteīna sekvences piesaistīšana pie AP205 apvalka proteīna un ekspresija E.coli šūnās nodrošināja mozaīkveida daļiņu veidošanos. Mozaīkveida daļiņas veidojās arī ģenētiski pievienojot HIV-Tat un WNV-DIII proteīnu sekvences pie GA apvalka proteīna un ekspresējot tās raugos. Abi WNV apvalka proteīnu saturošie nesēji parādīja specifisku imūno atbildi. Otrkārt, tika novērota IL-2 un GFP mRNS in vivo iepakošana GA apvalka proteīna veidotajās VLD, sintezējot tās S.cerevisiae. Iepakošanās tika uzlabota, papildinot mRNS ar MS2 operatora sekvenci un izmantojot GA VLD mutantus ar MS2 operatoram līdzīgu operatora saitu. Treškārt, metodika tukšu pilna garuma HBV core daļiņu iegūšanai tika izstrādāta un veiksmīgi pielietota daļiņām, kas ekspresētas baktērijās un raugos. Četri jauni HBV core proteīni (HBc-K75, HBc-K77, HBc-K79, HBc-K80) ar lizīna punktveida mutācijām uz daļiņu ārējās virsmas var tikt izmantoti perspektīvai adresēšanai, izmantojot ķīmisko piešūšanu. Mēs pārbaudījām, ka izstrādātā sārmainās apstrādes metode ir efektīva visu sešu veidu – bakteriālās izcelsmes, rauga izcelsmes un četru lizīna mutantu – tukšo daļiņu iegūšanai. Ceturtkārt, tika pētīta nukleīnskābju fragmentu in vitro iepakošana ar sārmu apstrādātajās tukšajās HBc VLD. DNS fragmentu iepakošanai tika izmantota daļiņu uzirdināšana urīnvielā un tai sekojoša rekonstrukcija, savukārt RNS un īsi oligodezoksinukleotīdi tika iekļauti VLD sastāvā tos vienkārši pievienojot. Tika parādīts, ka individuālie divpavediena DNS fragmenti ar maksimālo garumu starp 1289 bp un 1737 bp ir spējīgi iepakoties daļiņās vienādās molārās attiecībās, savukārt garāki DNS fragmenti un plazmīdas tika ieskautas ar tukšajām daļiņās to lielā molārā pārākumā. Iepakotās DNS bija pasargātas no DNāzes, turpretim RNS gadījumā, neskatoties uz tā spēju piesaistīties un iepakoties tukšās VLD, tik novērota spēcīga izekstraģētā materiāla degradācija, ko varētu skaidrot ar iespējamu tukšo HBV core VLD ribonukleāžu aktivitāti. Mūsu jaunās un pespektīvās tehnoloģijas mazo RNS fāgu un HBV core pētījumu jomā var tikt izmantotas vakcīnu izstrādei vai arī pielietotas piegādes vektoru izveidei ar spēju tikt ķīmiski adresētām specifiskai mijiedarbībai ar šūnām. Atslēgvārdi: VLD, core, iepakošanaVirus-like particles (VLPs) are protein structures formed from one or few types of protein capable to self-assemble in complex and highly organised structures. Due to the symmetrical fashion and multimeric structure pattern, VLPs can be used in two ways – as perspective carriers for exposition of foreign sequences and as a delivery vehicles containing packaged cargo inside them. One of the most promising area of VLPs utilization is vaccine development. Bacteriophages GA and AP205 are among one of the smallest and simplest viruses, extensively used as a study model for molecular biology. Coat protein expressed in bacteria or yeast forms VLPs. Another object – hepatitis B virus (HBV) core protein produced as recombinant protein also self-assemble into VLPs. The aim of the work was to test various virus-like particle usage possibilities, therefore different test objects were used. First, West-Nile virus protein E domain III (WNV-DIII) protein sequence attachment to AP205 coat protein leaded to mosaic particle formation when expressed in E.coli cells. Mosaic particle formation was also achieved when HIV-Tat or WNV-DIII protein sequences were genetically attached to phage GA coat protein and expressed in yeast. Both WNV envelope protein equipped carriers showed specific immune response. Second, in vivo packaging of IL-2 and GFP mRNA into GA coat protein particles synthesized in S.cerevisiae was achieved. The packaging was improved by supplementing MS2 operator sequence to mRNA and using GA VLP mutants that mimicks MS2 operator binding site. Third, the methodology to obtain empty full-length HBV core VLPs was elaborated and successfully used for both bacteria- and yeast-produced particles. Four novel HBV core proteins (HBc-K75, HBc-K77, HBc-K79, HBc-K80) with lysine point-mutations on the protruding structure elements of particles, reveal perspective for addressing through chemical coupling. We proved, that elaborated alkali treatment method is effective for obtaining empty particles for all six cases – yeast-source, bacteria-source, as well for four lysine mutants. Fourth, in vitro packaging of nucleic acid fragments into alkali-treated empty HBc VLPs was studied. Packaging of DNA fragments was achieved through scarification of particles in urea and subsequent reconstruction, whereas RNA and short oligodeoxynucleotides were included in VLPs by simple mixing. Individual double-stranded DNA fragments of maximum length in range between 1289 to 1737 base pairs were found as packaged in equimolar ratio, whereas longer DNA fragments / plasmids were embraced by empty particles in large molar excess of them. Packaged DNA was protected from DNAse, whereas RNA, despite its capability to bind and package into VLPs, after extraction showed strong degradation, possibly explainable with ribonuclease activity of empty HBc VLPs. Our novel and perspective technologies in field of small RNA phage and HBv core based virus-like particles can be used for vaccine development or utilized for generation of delivery cargo with abilities te be chemically addressed for specifical targeting and interaction with cells of interest. Keywords: VLP, core, packagin

Immunization of Cats against Fel d 1 Results in Reduced Allergic Symptoms of Owners.

An innovative approach was tested to treat cat allergy in humans by vaccinating cats with Fel-CuMV (HypoCatTM), a vaccine against the major cat allergen Fel d 1 based on virus-like particles derived from cucumber mosaic virus (CuMV-VLPs). Upon vaccination, cats develop neutralizing antibodies against the allergen Fel d 1, which reduces the level of reactive allergen, thus lowering the symptoms or even preventing allergic reactions in humans. The combined methodological field study included ten cat-allergic participants who lived together with their cats (n = 13), that were immunized with Fel-CuMV. The aim was to determine methods for measuring a change in allergic symptoms. A home-based provocation test (petting time and organ specific symptom score (OSSS)) and a general weekly (or monthly) symptom score (G(W)SS) were used to assess changes in allergic symptoms. The petting time until a pre-defined level of allergic symptoms was reached increased already early after vaccination of the cats and was apparent over the course of the study. In addition, the OSSS after provocation and G(W)SS recorded a persistent reduction in symptoms over the study period and could serve for long-term assessment. Hence, the immunization of cats with HypoCatTM (Fel-CuMV) may have a positive impact on the cat allergy of the owner, and changes could be assessed by the provocation test as well as G(W)SS

Organ-On-A-Chip (OOC) Image Dataset for Machine Learning and Tissue Model Evaluation

Organ-on-a-chip (OOC) technology has emerged as a groundbreaking approach for emulating the physiological environment, revolutionizing biomedical research, drug development, and personalized medicine. OOC platforms offer more physiologically relevant microenvironments, enabling real-time monitoring of tissue, to develop functional tissue models. Imaging methods are the most common approach for daily monitoring of tissue development. Image-based machine learning serves as a valuable tool for enhancing and monitoring OOC models in real-time. This involves the classification of images generated through microscopy contributing to the refinement of model performance. This paper presents an image dataset, containing cell images generated from OOC setup with different cell types. There are 3072 images generated by an automated brightfield microscopy setup. For some images, parameters such as cell type, seeding density, time after seeding and flow rate are provided. These parameters along with predefined criteria can contribute to the evaluation of image quality and identification of potential artifacts. This dataset can be used as a basis for training machine learning classifiers for automated data analysis generated from an OOC setup providing more reliable tissue models, automated decision-making processes within the OOC framework and efficient research in the future